Download A Novel Small-Molecule Inhibitor of Transforming Growth Factor B

Research Article
A Novel Small-Molecule Inhibitor of Transforming Growth
Factor B Type I Receptor Kinase (SM16) Inhibits Murine
Mesothelioma Tumor Growth In vivo and Prevents
Tumor Recurrence after Surgical Resection
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Eiji Suzuki, Samuel Kim, H.-Kam Cheung, Michael J. Corbley, Xiamei Zhang,
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Lihong Sun, Feng Shan, Juswinder Singh, Wen-Cherng Lee,
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Steven M. Albelda, and Leona E. Ling
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Thoracic Oncology Research Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania and 2Oncology Cell Signaling,
Biogen Idec, Cambridge, Massachusetts
Abstract
Malignant mesothelioma is an aggressive and lethal pleural
cancer that overexpresses transforming growth factor B
(TGFB). We investigated the efficacy of a novel small-molecule
TGFB type I receptor (ALK5) kinase inhibitor, SM16, in the
AB12 syngeneic model of malignant mesothelioma. SM16
inhibited TGFB signaling seen as decreased phosphorylated
Smad2/3 levels in cultured AB12 cells (IC50, f200 nmol/L).
SM16 penetrated tumor cells in vivo, suppressing tumor
phosphorylated Smad2/3 levels for at least 3 h following
treatment of tumor-bearing mice with a single i.p. bolus of
20 mg/kg SM16. The growth of established AB12 tumors was
significantly inhibited by 5 mg/kg/d SM16 (P < 0.001) delivered
via s.c. miniosmotic pumps over 28 days. The efficacy of SM16
was a result of a CD8+ antitumor response because (a) the antitumor effects were markedly diminished in severe combined
immunodeficient mice and (b) CD8+ T cells isolated from
spleens of mice treated with SM16 showed strong antitumor
cytolytic effects whereas CD8+ T cells isolated from spleens of
tumor-bearing mice treated with control vehicle showed
minimal activity. Treatment of mice bearing large tumors with
5 mg/kg/d SM16 after debulking surgery reduced the extent of
tumor recurrence from 80% to <20% (P < 0.05). SM16 was also
highly effective in blocking and regressing tumors when given
p.o. at doses of 0.45 or 0.65 g/kg in mouse chow. Thus, SM16
shows potent activity against established AB12 malignant
mesothelioma tumors using an immune-mediated mechanism
and can significantly prevent tumor recurrence after resection
of bulky AB12 malignant mesothelioma tumors. These data
suggest that ALK5 inhibitors, such as SM16, offer significant
potential for the treatment of malignant mesothelioma and
possibly other cancers. [Cancer Res 2007;67(5):2351–9]
Introduction
Transforming growth factor h (TGFh) is a multifunctional
cytokine overexpressed by a variety of tumors (1–3). Many human
cancers show a correlation between overexpression of TGFh and
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Steven M. Albelda, Thoracic Oncology Research Laboratory,
University of Pennsylvania, BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104.
Phone: 215-573-9969; Fax: 215-573-4469; E-mail: [email protected].
I2007 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-2389
www.aacrjournals.org
advanced disease or poor prognosis (1–3). Neutralization of
TGFh has shown potential clinical utility in a variety of murine
models of cancer, including breast cancer (4–8), thymoma (9),
hepatocellular carcinoma (10), glioma (11, 12), head and neck
carcinoma (13), and malignant mesothelioma (14, 15). Depending on the model, the antitumor activity of TGFh antagonists is
mediated by one or more autocrine or paracrine mechanisms,
including increased immune surveillance, inhibition of angiogenesis, inhibition of invasion, metastases and epithelial to
mesenchymal transition, as well as the inhibition of collagen
deposition and tumor interstitial pressure (1, 2, 16–18).
Malignant mesothelioma is an aggressive pulmonary cancer of
the pleura and serosa, which has been shown to overexpress
TGFh (reviewed in ref. 15). Pleural fluids from mesothelioma
patients contain TGFh levels that are 3- to 6-fold elevated
compared with primary lung cancers (19, 20). Several mesothelioma cell lines also overexpress TGFh (reviewed in ref. 15). We
showed previously that TGFh seems to contribute significantly to
the growth of TGFh1-overexpressing malignant mesothelioma
cells in murine malignant mesothelioma tumor models (15).
Treatment of tumor-bearing mice with the soluble TGFh type II
receptor fusion protein (sTGFhRII:Fc) inhibited the growth of
tumors in two established syngeneic malignant mesothelioma
tumor models, AB12 and AC29. These effects seemed to be
primarily through the induction of CD8+ T-cell antitumor activity.
These preclinical results in malignant mesothelioma and other
cancer models show the potential for TGFh blocking agents in
anticancer therapy.
Several TGFh antagonist agents have been developed for
preclinical and clinical investigation. In addition to sTGFhRII:Fc,
an agent that binds TGFh1 and TGFh3 with high affinity, several
monoclonal antibodies (mAb) and antisense oligonucleotide agents
that bind various isoforms of TGFh have been developed. Of these,
one TGFh1 antisense oligonucleotide is in clinical development in
oncology and has shown promising results in a phase I glioma trial
(21). Additional TGFh antagonist agents are in preclinical and
clinical development (1).
Small-molecule inhibitors of TGFh type I receptor (ALK5) kinase
have also been developed recently (16, 22). Therapeutically, smallmolecule kinase inhibitors provide increased flexibility in dosing
regimen and route of administration (i.e., p.o. delivery) compared
with both protein- and oligonucleotide-based TGFh antagonist
agents. They also lack the potential for inducing neutralizing
antibodies inherent in the protein-based TGFh antagonist agents.
Several of these ALK5 kinase inhibitors have shown efficacy in
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models of fibrosis (23–25).3 However, the efficacy of ALK5
inhibitors in preventing tumor growth was only recently shown
in an orthotopic model of glioma (12), and several recent reports
suggest activity in breast, head and neck, and lung cancer tumor
models (13, 26, 27).
To determine if ALK5 kinase inhibitors are effective in treating
malignant mesothelioma, we used a novel ALK5 inhibitor, SM16
(13).3 This small-molecule kinase inhibitor, like those previously
described (28), was shown to be a potent selective inhibitor of
ALK5 and ALK4, the activin type IB receptor. Our results using SM16
delivered via osmotic pumps or by p.o. administration show that this
small, p.o. available inhibitor of TGFh signaling can inhibit the
growth of and even eradicate established non–TGFh-responsive
tumors without overt toxicity by inhibiting the immunosuppressive
activities of TGFh. Agents, such as SM16, show promise for difficult
to treat tumors, such as malignant mesothelioma.
Materials and Methods
Animals
Pathogen-free female BALB/c mice (6–8 weeks old, 19–24 g) were
purchased from Taconic Laboratories (Germantown, NY). CB-17 severe
combined immunodeficient (SCID) mice (6–8 weeks old, 19–24 g) were bred
at the Wistar Institute (Philadelphia, PA). All mice were maintained in a
pathogen-free animal facility for at least 1 week before each experiment.
The animal use committees of the Wistar Institute and University of
Pennsylvania approved all animal study protocols described in this
publication, and experiments were conducted in compliance with the
Guide for the Care and Use of Laboratory Animals.
Cell Lines
A murine malignant mesothelioma cell line, AB12, was obtained from Dr.
Bruce Robinson (University of Western Australia, Nedlands, Western
Australia, Australia). This line was derived in BALB/c mice and grows well
as flank tumors (29). AB12 cells secrete large amounts (462 pmol/106 cells/
24 h) of TGFh, with most of this TGFh in latent form (30). L1C2 cells are a
mouse lung cancer cell line obtained from the American Type Culture
Collection (Rockville, MD). Both cell lines were cultured and maintained in
high-glucose DMEM (Mediatech, Washington, DC) supplemented with 10%
fetal bovine serum (FBS; Georgia Biotechnology, Atlanta, GA), 100 units/mL
penicillin, 100 Ag/mL streptomycin, and 2 mmol/L glutamine. The cell line
was regularly tested and maintained negative for Mycoplasma spp.
ALK5 Inhibitor (SM16)
SM16, an ALK5/ALK4 kinase inhibitor with a molecular weight of 430,
was described previously (13).3 Briefly, SM16 binds ALK5 (K i, 10 nmol/L)
and ALK4 (K i, 1.5 nmol/L) with high affinity at the ATP-binding site. In
HepG2 cells, SM16 inhibits TGFh-induced plasminogen activator inhibitorluciferase activity (IC50, 64 nmol/L) and TGFh- or activin-induced Smad2
phosphorylation at concentrations between 100 and 620 nmol/L. SM16 was
tested against >60 related and unrelated kinases and showed moderate offtarget activity only against Raf (IC50, 1 Amol/L) and p38/SAPKa (IC50,
0.8 Amol/L). SM16 exhibited no inhibitory activity against ALK family
members ALK1 and ALK6.3 ALK7 blocking activity has not been determined.
Inhibition of TGFB-Induced Smad2/3 Phosphorylation in
AB12 Cells
Confluent AB12 cells were preincubated for 1 h at 37jC in 2 mL DMEM
plus 0.5% FBS supplemented with a 1:3 dilution series of SM16. Final DMSO
concentration was 1%. TGFh1 (R&D Systems, Minneapolis, MN) was added
and cells were incubated for 1.5 h at 37jC. The cells were rinsed with cold
3
K. Fu, et al. An orally active inhibitor of the TGF-h type I receptor, ALK5, inhibits
vascular fibrosis and adventitial myofibroblast induction in the rat carotid balloon
injury model, submitted for publication.
Cancer Res 2007; 67: (5). March 1, 2007
PBS and lysed in 150 AL SDS-PAGE loading buffer containing 20 mmol/L NaF
and a protease inhibitor cocktail (‘‘Complete,’’ Roche, Nutley, NJ). The samples
were separated on a 10% SDS-PAGE gel and transferred to nitrocellulose
membranes for Western blot analysis. The membranes were blocked for 1 h at
room temperature in 5% milk/PBS-Tween 20. Total Smad2 was analyzed with
a rabbit polyclonal anti-Smad2/3 primary antibody (Cell Signaling, Beverly,
MA), 1:2,000 in 5% milk, overnight at 4jC followed by a horseradish
peroxidase (HRP)-linked goat anti-rabbit secondary antibody (Bio-Rad,
Hercules, CA), 1:4,000, for 1 h at room temperature. The blot was stripped
with Restore (Pierce Biotechnology, Rockford, IL). Phosphorylation of Smad2
was analyzed with a rabbit polyclonal anti–phosphorylated Smad2 primary
antibody (Cell Signaling), 1:2,000 in 5% milk, overnight at 4jC followed by the
same secondary antibody. Blots were developed with SuperSignal West Pico
(Pierce Biotechnology) and visualized by exposure to film.
Inhibition of TGFB Signaling in AB12 Tumors In vivo by
SM16
BALB/c mice were injected on the right flank with 1 106 AB12 tumor
cells. When tumors reached 200 to 250 mm3 in size, the mice were injected
with a single i.p. bolus of 40 mg/kg SM16 in 20% Captisol (CyDex, Inc.,
Lenexa, KS). The mice were sacrificed and the AB12 tumors were dissected
and snap frozen in liquid nitrogen. To measure phosphorylated Smad2 and
total Smad2 in tumors, pulverized frozen AB12 tumor samples were
extracted for 1 h at 4jC in T-PER buffer (Pierce Biotechnology) containing
protease inhibitors (Roche). A total of 5 Ag of each tumor extract was
separated on SDS-PAGE and transferred to nitrocellulose membrane for
Western blot analysis. Phosphorylated Smad2/3 levels were detected as
above. Total Smad2 protein levels were detected by stripping and reprobing
the blots or by probing duplicate blots with an anti-Smad2 mouse primary
antibody at 0.1 Ag/mL (Transduction Laboratories, Lexington, KY) and a
goat anti-mouse IgG HRP secondary antibody at 1:100,000 dilution (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA), and the blot was then
developed as above.
AB12 Tumor Efficacy Models
Antitumor efficacy with SM16 delivered via osmotic pump. BALB/c
mice were injected on the right flank with 1 106 AB12 tumor cells. For s.c.
pump delivery, SM16 was formulated in vehicle, 20% Captisol, at various
concentrations (0, 5, 10, and 20 mg/mL) and loaded into Alzet model 2004
miniosmotic pumps (Alzet Corp., Cupertino, CA). The pumps were allowed
to prime in sterile saline for 18 to 20 h before implantation to reach the
expected dispensing rate of 0.25 AL/h. At this rate, the above concentrations
of SM16 would provide doses of 0, 1.25, 2.5, and 5 mg/kg/d per 23 g mouse.
When the tumors reached a minimal volume of 300 mm3 (f12 days after
tumor cell inoculation), mice were anesthetized with i.p. injection of 70 mg/
kg ketamine and 7 mg/kg xylazine and the pumps were implanted s.c. on
the left flank of the mice. The SCID mice were studied using the same
experimental design as the BALB/c mice. Tumor volumes were estimated
using the formula (k long axis short axis short axis) / 6. We did
measurements of tumors twice weekly. At sacrifice, plasma was obtained
under anesthesia and analyzed for SM16 plasma levels.
Debulking surgery models. BALB/c mice were injected on the right
flank with 1 106 AB12 tumor cells. When the tumors reached a minimal
volume of 850 mm3 (f25 days after tumor cell inoculation), mice were
anesthetized and a complete resection of the tumors was attempted. All
macroscopically visible tumor was removed and the wound was closed
using silk suture. At the time of debulking, mice were randomly divided into
two groups and one group was implanted with minipumps loaded with 20%
Captisol (control) on the left flank and the other group was implanted with
minipumps loaded with 20 mg/mL SM16. Tumor recurrence was defined as
the first day when a tumor was unambiguously visible or palpable
(approximately 2 2 mm). Unless otherwise mentioned, each control or
experimental group had a minimum of five mice. Plasma was obtained
under anesthesia and analyzed for SM16.
P.o. delivery model. AB12 cells were injected into the right flanks of
BALB/c mice as described above. Mice were fed standard chow ad libitum.
When tumors grew to f100 mm3, the mice were randomized into
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TGF-b Inhibitor Blocks Malignant Mesothelioma Growth
treatment groups and fed either standard chow or chow formulated with
SM16 at the indicated concentrations (Research Diets, New Brunswick, NJ).
Tumor growth and animal weights were monitored every 3 days. Tumor
volumes were determined as described above. Chow consumption was
monitored every 4 to 5 days, and behavioral observations were monitored
daily. At the conclusion of the study, plasma was obtained under anesthesia
and analyzed for SM16.
In vivo Tumor Neutralization Assay (Winn Assay)
Winn assays were done as described previously (31). Splenocytes were
isolated and CD8+ T lymphocytes were purified using the MACS system
(Miltenyi Biotec, Auburn, CA). This cell population contained >90% CD8+
cells by flow cytometry (data not shown). The CD8+ T lymphocyte–enriched
population from normal, tumor-sensitized, or tumor-sensitized and treated
mice was admixed with viable AB12 tumor cells at a ratio of three purified
CD8+ splenocytes for each tumor cell, and the mixture was inoculated s.c.
into the flanks of naive BALB/c mice. Each mixture thus contained 0.5 106
tumor cells and 1.5 106 CD8+ T cells. This ratio has previously been
determined to be optimal for detecting positive and negative effects (32). To
show specificity, Winn assays were also done as above but using L1C2 lung
cancer cells (which grow in BALB/c mice) as targets. Tumor growth was
measured after 1 week and expressed as the mean F SE of at least five mice
per group.
Bioanalytic Analysis of SM16 in Plasma
The concentration of SM16 in plasma was analyzed by high-performance
liquid chromatography on a Zorbax SB-C8 3.5-Am (2.1 50 mm) column
(Agilent, Palo Alto, CA) followed by mass spectrometry (MS) on a triple
quadrupole mass spectrometer (SCIEX API 4000, Applied Biosystems, Foster
City, CA) equipped with a Turbo Ion Spray probe operated in positive ion
mode. Plasma SM16 was first subjected to solid-phase extraction on an
Oasis HLB AElution SPE plate (Waters, Milford, MA) preconditioned with
methanol and water (SPE), washed with 5% methanol, and eluted with
acetonitrile/isopropanol (40:60). Samples were further diluted with 50:50:0.1
acetonitrile/water/formic acid (v/v/v) before analysis by liquid chromatography-MS/MS using multiple reaction monitoring. Data were collected and
processed using Analyst version 1.4.1 (Applied Biosystems).
Statistical Analyses
Data comparing differences between two groups were assessed using
unpaired Student’s t test. ANOVA with post hoc testing was used for
multiple comparisons. Differences in survival were analyzed using the logrank test. Differences were considered significant when P was <0.05.
Statistical analysis was conducted using the StatView 5.0 for Windows
program.
Results
Inhibition of TGFB signaling in cultured AB12 cells and
AB12 tumors by SM16. The ability of SM16 to inhibit TGFh
signaling was evaluated in AB12 cells in vitro. As previously reported (15), AB12 cells express a low basal level of phosphorylated
Smad2, which is further increased by incubation with exogenous
TGFh (Fig. 1A, left). However, addition of TGFh (10 ng/mL) along
with SM16 at concentrations ranging from 8 to 2,000 nmol/L
prevented the TGFh-dependent elevation of phosphorylated
Smad2 in a dose-dependent manner. The total amount of Smad2
protein was unchanged under these conditions. The approximate
IC50 for inhibition of phosphorylated Smad2 in these cells was
200 nmol/L.
AB12 tumors, formed after s.c. implantation of cells in syngeneic
BALB/c mice, showed constitutive activation of the TGFh pathway
as indicated by the presence of phosphorylated Smad2 in these
tumors (Fig. 1B , vehicle treated). Administration of SM16
significantly decreased the phosphorylated Smad2 level in these
tumors for up to 12 h, with the phosphorylated Smad2 signal
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Figure 1. Inhibition of TGFh signaling (Smad2 phosphorylation) in cultured
AB12 cells and AB12 tumors by SM16. A, AB12 cells in culture were exposed
to varying concentrations of SM16 followed by 10 ng/mL TGFh. After 1.5 h,
protein lysates were prepared and equal amounts of protein were subjected to
Western blot analysis with an anti–phosphorylated Smad2 (P-Smad2) antibody
and then stripped and reanalyzed with an anti-Smad2 antibody. Smad2 was
recognized as a band at a molecular weight of 52 to 55 kDa. SM16 added at
various concentrations, along with TGFh, prevented the TGFh-dependent
elevation of phosphorylated Smad2 in a dose-dependent manner. B, mice
bearing AB12 flank tumors were treated i.p. with vehicle or 40 mg/kg SM16
(Tumors ). At various times after injection, tumors were removed and protein
lysates were prepared for Western blot analysis as in (A ). I.p. administration of
SM16 significantly decreased the phosphorylated Smad2 levels in these tumors
up to 12 h after administration. Lysates of AB12 cells (Cells ), which are
unstimulated () or stimulated (+) for 1.5 h with 10 ng/mL TGFh, were also
analyzed as controls for the presence of phosphorylated Smad2. Smad2 was
recognized as a band at a molecular weight of 52 to 55 kDa.
returning at 24 h after administration (Fig. 1B). These results
suggest that SM16 is able to distribute to s.c. AB12 tumors and
inhibit TGFh signaling in these tumors for at least 12 h after a
single dose.
SM16 is an effective inhibitor of AB12 tumor growth. To
investigate the potential therapeutic utility of SM16 and to
determine the circulating level of SM16 required for efficacy, we
gave various concentrations of SM16 using miniosmotic pumps to
mice bearing established AB12 tumors. AB12 tumors in BALB/c
mice were allowed to reach 300 mm3 in size, at which time
treatment with SM16 was initiated. No differences were observed in
tumor growth in mice that did not undergo surgical pump
implantation compared with those surgically implanted with
pumps containing the vehicle alone (data not shown). However,
SM16 significantly inhibited tumor growth at a dose of 5 mg/kg/d
compared with the vehicle control (P < 0.001; Fig. 2A). At day 28,
tumors treated with 5 mg/kg/d SM16 were 599 F 59 mm3 versus
1,358 F 127 mm3 in control groups, a more than 2.4-fold reduction
in size. Treatment with the two lower doses, 1.25 and 2.5 mg/kg/d,
showed a nonstatistical trend of tumor growth inhibition
compared with vehicle control, suggesting dose-dependent inhibition by SM16.
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Figure 2. SM16 is an effective inhibitor of AB12 tumor growth. A, AB12 tumors
in BALB/c mice were allowed to reach 300 mm3 in size, at which time treatment
with SM16 was initiated (day 17). Tumor size was measured every 3 d. SM16
significantly inhibited tumor growth at 5 mg/kg/d dose compared with the
vehicle control. *, P < 0.001. B, bioanalytic analysis of SM16 levels in plasma
at day 28 at various doses of drug given by miniosmotic pump.
SM16 levels in plasma at day 28 were measured to identify the
circulating SM16 concentration required to achieve efficacy.
Figure 2B shows that the average plasma concentration of SM16
was 0.77 F 0.08 Amol/L at 1.25 mg/kg/d, 0.70 F 0.07 Amol/L at
2.5 mg/kg/d, and 1.52 F 0.33 Amol/L at 5 mg/kg/d. Statistically
significant efficacy was achieved at the 5 mg/kg/d dose, which
corresponded to a plasma concentration of 1.5 Amol/L. Interestingly, a similar concentration (2 Amol/L) of SM16 was able to
significantly inhibit phosphorylated Smad2 in cultured AB12 cells
to unstimulated levels (Fig. 1A). These data show the efficacy of
SM16 in inhibiting the growth of AB12 malignant mesothelioma
tumors in this syngeneic model.
SM16 efficacy is primarily immune cell mediated: loss of
efficacy in SCID mice. We next tested whether the efficacy of SM16
was primarily due to increased antitumor immune responses as
seen previously with sTGFhRII:Fc in this model (15). SCID mice
bearing AB12 tumors (300 mm3 in size) were treated with either
vehicle alone (control) or 5 mg/kg/d SM16. As shown in Fig. 3, there
was no significant difference in tumor volume between controltreated mice and SM16-treated mice at any time during the 12 days
of treatment. AB12 tumors grew significantly faster in SCID compared with BALB/c mice so that treatment began at day 7 and sacrifice of the SM16- and vehicle-treated groups was required 12 days
after the initiation of treatment. These results confirm the importance of lymphocytes for SM16 activity in the AB12 tumor model.
SM16 prevents loss of CTL activity. In our previous study, we
showed that (a) BALB/c mice bearing small AB12 tumors generate
Cancer Res 2007; 67: (5). March 1, 2007
endogenous CTL activity in the spleen; (b) when tumors became
larger in size, the endogenous CTL lost their ability to lyse target
AB12 tumors; and (c) blockade of TGFh using sTGFhRII:Fc in mice
bearing small AB12 tumors prevented the loss of CTL activity (15).
To evaluate if SM16 treatment also prevented the loss of CTL
activity in the AB12 tumor model, we assayed for splenic CTLs
using an in vivo tumor neutralization assay (Winn assay; Fig. 4).
We prepared two control groups of CD8+ T-cell preparations as
follows: group 1, naive CD8+ T cells from the spleens of nontumorbearing animals (negative control), and group 2, CD8+ T cells from
animals bearing ‘‘small’’ (f100 mm3, 7 days after tumor cell
inoculation) AB12 tumors (positive control). We also prepared two
experimental groups: group 3, CD8+ T cells from tumor-bearing
mice treated with vehicle alone, and group 4, CD8+ T cells from
tumor-bearing mice treated with 5 mg/kg/d SM16. In groups 3 and
4, animals with established AB12 tumors (300 mm3) were treated
with vehicle- or SM16-loaded pumps (SM16) for 10 days. After the
treatment for 10 days, the tumors grew to a size of f500 mm3 in
the control animals, whereas they were reduced to f200 mm3 in
treated animals. Splenocytes were isolated and CD8+ T cells were
purified from these two groups of animals.
CD8+ T cells from all groups were mixed with fresh AB12 tumor
cells in a ratio of three CD8+ T cells to one tumor cell, and the
mixtures were injected into the flanks of naive BALB/c mice. This
ratio was established from preliminary dose titration studies,
establishing that both positive and negative effects could be seen at
this ratio. Tumors were allowed to grow for 1 week. As shown in
Fig. 4, control AB12 tumor cells grew to a size of f300 mm3 in
1 week. Addition of CD8+ T cells from nontumor-bearing animals
(group 1) did not significantly slow the tumor growth. In contrast,
tumor cells mixed with CD8+ T cells from animals bearing ‘‘small’’
tumors (group 2) at a lymphocyte to tumor ratio of 3:1 grew to only
99 mm3. Compared with tumors mixed with naive CD8+ T cells
(group 1), this represented a 67% decrease of the growth (P < 0.01).
Thus, in animals bearing small tumors, some spontaneous
antitumor CD8+ T-cell activity is induced by the AB12 tumor cells.
As previously reported (15), as the tumor increases in size, this
CD8+ T-cell activity is lost. This can be seen in the T cells from
group 3, where CD8+ T cells from animals treated with vehicle
lost all antitumor activity. However, the mice treated with SM16
Figure 3. SM16 efficacy is primarily immune cell mediated: loss of efficacy in
SCID mice. SCID mice bearing AB12 tumors (300 mm3 in size) were treated with
either vehicle alone (control) or 5 mg/kg/d SM16 via minipump starting on day 7.
There was no significant difference in tumor volume between control-treated
mice and SM16-treated mice at any time during the treatment.
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Figure 4. SM16 prevents loss of CTL activity. A, splenic CTL activity was
measured using an in vivo tumor neutralization assay (Winn assay). CD8+ T cells
from the spleens of following groups were mixed with AB12 tumor cells and
injected into the flanks of naive mice. Tumor size was measured after 1 wk.
Groups were as follows: no CD8 cells added (AB12 cells only), naive CD8+
T cells from nontumor-bearing mice (group 1), CD8+ T cells from animals bearing
‘‘small’’ tumors (i.e., f100 mm3) 7 d after tumor cell inoculation (group 2),
CD8+ T cells from tumor-bearing mice that had been treated with vehicle alone
(group 3), and CD8+ T cells from tumor-bearing mice treated with SM16
(group 4). Control AB12 tumor cells grew to a size of f300 mm3 in 1 wk.
Addition of CD8+ T cells from nontumor-bearing animals (naive, group 1) did not
significantly slow the tumor growth. In contrast, tumor cells mixed with CD8+
T cells from animals bearing ‘‘small’’ tumors (group 2) at a lymphocyte to tumor
ratio of 3:1 grew to only 99 mm3. *, P < 0.01. However, CD8+ T cells from animals
bearing ‘‘large’’ tumors similar to group 1, but treated with vehicle (group 3),
lost all antitumor activity. **, significantly different from small AB12 tumors and
SM16-treated tumors. In contrast, mice treated with SM16 (SM16 CD8+ T cells,
group 4) showed persistent antitumor cytolytic activity. x, P < 0.01, compared
with AB12 and control CD8+ tumors. B, CD8+ cells from mice fed with SM16
chow had strong cytotoxic activity against AB12 cells. Extent of tumor growth in
mice 1 wk after implantation with AB12 cells alone (AB12 alone ), AB12 cells
mixed with splenic CD8+ T cells from nontumor-bearing mice (CD8/Non tumor
bearing ), AB12 cells mixed with splenic CD8+ T cells from AB12-bearing mice
(CD8/AB12 tumor bearing ), and AB12 cells mixed with splenic CD8+ T cells from
AB12-bearing mice that had been treated with SM16 (CD8/SM16 treated AB12
tumor bearing ). C, in contrast, CD8+ cells from mice fed with SM16 chow
showed no cytotoxic activity against L1C2 cells. Extent of tumor growth in mice
1 wk after implantation with L1C2 cells alone (L1C2 alone ), L1C2 cells mixed
with splenic CD8+ T cells from AB12-bearing mice (CD8/AB12 tumor bearing ),
and L1C2 cells mixed with splenic CD8+ T cells from AB12-bearing mice that had
been treated with SM16 (CD8/SM16 treated AB12 tumor bearing ).
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(group 4) showed persistent antitumor cytolytic activity. The
growth of tumor cells was inhibited by 77% (P < 0.01) compared
with the tumor volume of the naive or vehicle groups. These data
indicate that SM16 treatment also prevents loss of CTL activity
similar to what we observed in sTGFhRII:Fc treatment (15).
To show specificity and to document a similar effect in SM16
chow-treated animals, similar experiments as above were done
using a second target cell line that grows in BALB/c mice, the
lung cancer cell line (L1C2). Splenic CD8+ T cells from AB12bearing mice fed with SM16 chow showed strong cytotoxic
activity against AB12 cells (Fig. 4B) but not against L1C2 tumor
cells (Fig. 4C).
Treatment with SM16 after surgery markedly reduces the
extent of tumor recurrence. The data above confirmed that
SM16 could be useful for treatment of large established AB12
tumors and that CD8+ T cells were generated (or maintained).
Next, we evaluated the potential therapeutic utility of SM16 in
another clinically relevant animal model: postsurgical adjuvant
therapy.
BALB/c mice were injected on the right flank with 1 106 AB12
tumor cells. When the tumors reached a minimal volume of
850 mm3 a complete resection of the tumors was done and mice
were randomly divided into two groups. One group was implanted
with vehicle-loaded pumps (control) and the other group was
implanted with pumps loaded with 5 mg/kg/d SM16. After
resection of large AB12 tumors, only 14% of control-treated mice
remained recurrence-free at 19 days after debulking surgery. In
contrast, 83% of SM16-treated mice remained recurrence-free
(P < 0.01; Fig. 5). These data show that adjuvant therapy with SM16
markedly reduces the extent of tumor recurrence after surgery.
P.o. administration of SM16 induces tumor regression. The
data above clearly show the efficacy of SM16 when delivered via a
s.c. pump. For clinical use, however, it would be much more
desirable to give drug via the p.o. route. To further explore efficacy
and drug exposure obtainable with SM16 via a p.o. route, SM16 was
formulated at various doses (0.25, 0.45, 0.65, and 1.7 g/kg chow)
into standard mouse chow. Given that the mice consumed f5 g
chow per day, the dose of SM16 at the 0.65 g/kg dose was estimated
to be 3.25 mg/d.
Figure 5. Treatment with SM16 after surgery markedly reduces the extent of
tumor recurrence. BALB/c mice were injected on the right flank with AB12 tumor
cells. When the tumors reached a minimal volume of 850 mm3 (f25 d after
tumor cell inoculation), a complete resection of the tumors was attempted.
One group was implanted with pumps loaded with vehicle (control; E) and the
other group was implanted with pumps loaded with SM16 (5). After resection
of large AB12 tumors, only 14% of control-treated mice remained recurrencefree at 19 d after debulking surgery. In contrast, 83% of SM16-treated mice
remained recurrence-free. *, P < 0.01.
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Sampling of the plasma drug levels was done at two time
points in the day, early morning (a.m.) and afternoon (p.m.),
corresponding to periods shortly after peak activity and feed
consumption (a.m.) and during the nadir of activity and feed
consumption (p.m.) for this nocturnal species. The plasma levels of
SM16 achieved for each dose did not vary more than 1.5- to 2-fold
between the a.m. and p.m. time points (Fig. 6B). Levels of 4.3 F 2.6
Amol/L, 6.7 F 2.4 Amol/L, and 9.8 F 2.8 Amol/L, respectively, were
measured for the 0.25, 0.45, and 0.65 g/kg doses. As expected, the
vehicle group showed no detectable SM16 above background.
These plasma levels achieved through p.o. administration were
approximately 3-, 4.5-, and 6.5-fold higher than those obtained with
the maximal s.c. miniosmotic pump administration (level of 1.5
Amol/L), which is consistent with the increased efficacy noted in
these p.o. studies. In spite of these higher plasma levels, no
differences were observed in weight gain, feed consumption, or
appearance and behavior of the 0.25, 0.45, and 0.65 g/kg SM16
chow-fed versus control chow-fed mice (Fig. 6B). In contrast,
animals fed the highest dose, 1.7 g/kg, showed clear evidence of
toxicity manifested by an ill-looking appearance and a loss of
weight (Fig. 6C). No changes apparent in consumption of feed were
noted between the control and SM16 containing chow-fed groups
(data not shown).
Discussion
Figure 6. P.o. administration of SM16 induces tumor regression. A, mice
bearing AB12 tumors were treated with various doses of SM16 formulated into
mouse chow beginning when the tumors reached a size of 100 mm3 (day 6)
and tumor sizes were measured. Tumor size was significantly smaller (P < 0.01)
in all doses of SM16 tested. B, bioanalytic measurement of SM16 levels in
plasma. Sampling of the plasma drug levels was done at two time points in the
day: early morning (a.m.) and afternoon (p.m.). A dose response was observed.
The plasma levels of SM16 achieved for each dose did not vary more than
1.5- to 2-fold between the a.m. and p.m. time points and were dose proportional.
These plasma levels achieved through p.o. administration were approximately
3-, 4.5-, and 6.5-fold higher than those obtained with the s.c. miniosmotic
pump administration (Fig. 2B ). C, to assess toxicity of the SM16 chow, animals
given various doses were weighed every 3 d. Animals fed the higher dose,
1.7 g/kg, showed clear evidence of toxicity manifested by an ill-looking
appearance and a loss of weight and were euthanized. There were no apparent
adverse effects of dosing with SM16 on weight gain or appearance at the lower
doses.
Mice bearing AB12 tumors of f110 mm3 were treated with
various doses of SM16-formulated chow. The animals were
observed for tumor size, gross toxicity, and weight loss. As shown
in Fig. 6A, there was a dramatic reduction in tumor size induced by
all doses of SM16 tested. Tumors were actually completely eradicated at the 0.65 and 0.45 g/kg doses; however, even at 0.25 g/kg,
there was a marked and statistically significant (P < 0.01) decrease in
tumor volume over the 2 weeks of treatment such that the final mean
tumor size in this group was 97 mm3 or 11% of the mean tumor size
in the control chow-treated animals.
Cancer Res 2007; 67: (5). March 1, 2007
Malignant mesothelioma is an aggressive cancer with a median
survival of 6 to 9 months and no effective therapies (33). We and
others have shown previously that both mouse models of
malignant mesothelioma and human malignant mesothelioma
exhibit immunosuppressive activity preventing effective antitumor
responses (15, 30, 32). Given that malignant mesothelioma cell lines
and human tumors express high levels of TGFh in tumor tissue and
in pleural effusion fluid, it is likely that this highly immunosuppressive, protumor cytokine plays an important role in promoting
immunosuppression of antitumor responses. The inhibition of
TGFh by the sTGFhRII:Fc fusion protein and anti-TGFh1 antisense
results in growth inhibition in mouse models of malignant
mesothelioma (14, 15). These results suggest that blockade of
TGFh signaling may be of therapeutic benefit in malignant
mesothelioma. Although TGFh has a complex role in the
establishment and progression of epithelial tumors (3, 7, 17, 34),
there are now many studies, including our own, showing that
blocking TGFh in established tumors can limit growth and
metastasis (see Introduction). Accordingly, several approaches,
including antibodies, soluble receptors, and antisense oligonucleotides, are being evaluated (reviewed in refs. 1, 16, 22).
The studies presented here characterize the antitumor activity of
an ALK5 kinase inhibitor, SM16. The ability of SM16 to inhibit
ALK5-dependent TGFh signaling was shown in AB12 mouse
mesothelioma cells where SM16 inhibited TGFh-induced phosphorylated Smad2. SM16 exhibits the ability to penetrate AB12 s.c.
tumors because it is able to cause sustained inhibition of AB12
tumor phosphorylated Smad2 for at least 3 h following an i.p.
administration. To determine the effect of SM16 on tumor growth,
SM16 was initially given via a s.c. miniosmotic pump to AB12
tumor-bearing mice. This mode of delivery maintains consistent
plasma levels of SM16 over the course of the study (data not
shown). The increase in plasma levels of SM16 between the low
doses (1.25 and 2.5 mg/kg/d) and the high dose (5 mg/kg/d)
correlated well with the significant efficacy achieved at 5 mg/kg/d
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TGF-b Inhibitor Blocks Malignant Mesothelioma Growth
compared with the marginal effect at the low doses. These results
suggest that the antitumor effect of SM16 is dependent on exposure
and can be achieved with consistent inhibition of TGFh signaling
over time. The plasma levels of SM16 achieved in these studies
suggested that efficacy required concentrations of 1.5 Amol/L
SM16. At this concentration, SM16 also has significant ALK4 and
some p38 and Raf inhibition activity in in vitro biochemical assays.
It is theoretically possible that the in vivo activity of SM16 is
derived from ALK4 inhibition or in part from p38 and/or Raf
inhibition. However, the extent of inhibition of tumor growth and
stimulation of antitumor CD8+ T cells of SM16 is very similar to
that seen with the soluble TGFhRII:Fc fusion protein, a TGFhspecific antagonist, indicating that the efficacy of SM16 maybe
solely due to TGFh signaling antagonism.
The p.o. activity of SM16 was shown by the inhibition of AB12
tumor growth when SM16 was given through feed. This method of
delivery, like that achieved with the s.c. miniosmotic pump, also
resulted in constant circulating levels of SM16 and sustained
suppression of tumor phosphorylated Smad2 (data not shown).
The plasma concentration of SM16 was not significantly different
between the a.m. and p.m. bleeds within each dose group, whereas
there was a linear dose-dependent increase in plasma SM16 when
the compound was given at 0.25 to 0.65 g/kg SM16 in feed (Fig. 6B).
The highest dose group showed a much greater variability in
plasma levels at both time points, which was likely due to the effect
of the toxicity observed in this dose group on drug metabolism
(Fig. 6B and C). Significant efficacy was obtained at all dose levels
(Fig. 6A), and the plasma levels of SM16 for all dose groups in this
p.o. study exceeded 2 Amol/L, with the lowest group (0.25 g/kg)
showing f5 Amol/L plasma levels. These findings are also very
consistent with target plasma level of 1.5 Amol/L required for
efficacy identified in the s.c. miniosmotic pump studies.
The efficacy of SM16 against AB12 malignant mesothelioma
tumors, like that of the sTGFhRII:Fc fusion protein, seems to be
primarily immune mediated, specifically through CD8+ antitumor T
cells. The efficacy seen in BALB/c mice was lost in SCID mice, which
lack T and B cells (Fig. 3). In addition, antitumor CTL activity was
maintained in SM16-treated mice bearing large AB12 tumors,
whereas the antitumor CTL activity found in all small AB12 tumorbearing mice was lost in vehicle-treated mice when their tumors
grew large (Fig. 4). In addition, tumors from control mice and animals treated with SM16 for 5 days were stained for apoptosis,
proliferation, and angiogenesis using the same methods and reagents as described by Ge et al. (26). Although we could see increased
areas of necrosis and inflammation in the SM16-treated tumors,
staining for apoptosis, proliferation, and angiogenesis was all quite
low and unchanged between treatment groups (data not shown).
The blockade of antitumor CTL activity by TGFh and the
enhancement of CTL activity by TGFh antagonists have been
documented in glioma (12), breast cancer (26), thymoma, and
prostate models (35–37). Indeed, the lack of clinical benefit
following immunotherapy-induced, systemic antitumor CTLs may
be due to the expression of immunosuppressive cytokines, such as
TGFh, in certain cancers (38–40). TGFh is a potent immunosuppressing cytokine and is found at high levels in pleural effusion
fluid from human mesothelioma patients (20). In mesothelioma
and other human cancers, antitumor CTLs are elicited but often
lack antitumor killing activity, remain at the periphery of tumors,
and exhibit markers of inactivation (39–43). It has been postulated
that these antitumor CTLs are inhibited by cytokines, such as
TGFh or interleukin-10, expressed by tumors or T regulatory cells
www.aacrjournals.org
resident in tumors (35, 39, 44). Indeed, recent studies show that the
TGFh may inhibit CTL activity through inhibition of granzyme
activity (36). Given these findings, TGFh antagonists, such as SM16,
may prove to be useful immunotherapy agents for the treatment of
malignant mesothelioma and other tumors dependent on TGFh for
immunosuppression.
Most forms of immunotherapy are maximally effective with
minimal tumor burdens. This results in a more optimal ratio of
CTL to tumor cells, allows better access of immunocytes to tumor
cells, and minimizes the local and systemic immunosuppression
induced by most tumors (45, 46). We hypothesized that one way to
achieve this more optimal situation would be to combine SM16
therapy with surgical debulking. In most human and animal
tumors, surgical debulking, by itself, is largely ineffective; there is
usually rapid regrowth of tumor. In the AB12 malignant
mesothelioma model, we also observed high recurrence rates
(86%) after debulking large flank tumors (Fig. 5; ref. 47). However,
when debulking was accompanied by SM16 therapy, the rate of
recurrence was markedly reduced to 17%. We hypothesize that
TGFh blockade prevented tumor-induced immunosuppression and
allowed effective antitumor immune responses to occur.
TGFh antagonists, including ALK5 inhibitors, have also shown
antimetastatic activity in mouse models of tumor metastases (6–8,
12, 27, 48, 49). This activity may be associated with the proinvasive,
angiogenic, prosurvival, and stromal-inducing activities of TGFh
(1). Indeed, TGFh is associated with tumor recurrence, metastases,
and poor prognosis in a significant number of human cancers
(1–3). Together, the immune-enhancing, anti-invasive, antiangiogenic, and antimetastatic activity of TGFh antagonists suggests
that agents, such as SM16, are particularly well suited for treatment
of cancer recurrence after debulking.
ALK5 inhibitors differ from the TGFh ligand-binding agents in
their ability to block both the TGFh and activin/nodal pathways
at the level of TGFh and activin type I receptors, ALK5 and ALK4
respectively. The ability to inhibit signaling by all isoforms of
TGFh differs from the selective TGFh1 and TGFh3 blockade
afforded by the sTGFhRII:Fc or selective TGFh1 or TGFh2 mAbs.
There is also some evidence that the activin signaling may
function similarly to TGFh in preventing epithelial hyperplasia
while increasing the malignancy of advanced cancers (50–52).
This suggests that activin, like TGFh, may play a protumorigenic
role especially in later stage cancers (53–57). Although this
additional activity of SM16 may be important in tumors where
activin/nodal signaling contributes to tumor growth, the efficacy
and mechanism of action obtained with SM16 and the
sTGFhRII:Fc were very similar, suggesting that TGFh may be
the primary target in the AB12 model of mesothelioma.
Our results with SM16 in mesothelioma are very consistent with
the findings in a recent publication of Ge et al. (26) who used a
different p.o. bioavailable TGFh type I receptor kinase inhibitor
(SD-208) to treat murine breast cancer. Like SM16, SD-208 was able
to (a) inhibit Smad2 phosphorylation in vitro and in vivo at similar
concentrations, (b) inhibit the growth of 4T1 breast cancer cells
in immunocompetent animal but not in nude mice, and (c) stimulate the generation of CTLs. Of note, treatment with SD-208 was
only attempted in animals with minimal disease; drug was given
only 1 day after tumor cell injection. In contrast, we used SM16 to
treat relatively large established tumors as well as minimal disease
(in the surgical adjuvant setting).
In summary, the studies presented here show the potential
utility of ALK5 inhibitor SM16 in treating malignant tumors,
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especially those expressing high levels of TGFh, such as malignant
mesothelioma. Although this article focused in detail on a
mesothelioma cell line, we have data that SM16 has similar
antitumor effects in other types of established tumors, including
pharyngeal carcinoma (13), colorectal carcinoma, lung cancer,
and thymoma (Supplementary Data).4 SM16 can effectively inhibit
TGFh signaling in vitro and in vivo and that this compound can
effectively reduce the growth of malignant mesothelioma in an
animal model of tumor growth without overt toxicity. The effects
in this system are primarily due to augmentation of immune
responses. The drug was effective when given by osmotic
minipump or p.o. In addition to inhibiting the growth of esta-
4
In preparation.
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Acknowledgments
Received 7/5/2006; revised 11/21/2006; accepted 12/14/2006.
Grant support: National Cancer Institute grant PO1 CA 66726.
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.
We thank Cheryl Black, Tracy Kruger, Van Phan, Liyu Yang, Ellen Rohde, and
Zhaoyang Li for their expertise in providing bioanalytic mass spectrometry analysis for
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Cancer Res 2007; 67: (5). March 1, 2007
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Research.
A Novel Small-Molecule Inhibitor of Transforming Growth
Factor β Type I Receptor Kinase (SM16) Inhibits Murine
Mesothelioma Tumor Growth In vivo and Prevents Tumor
Recurrence after Surgical Resection
Eiji Suzuki, Samuel Kim, H.-Kam Cheung, et al.
Cancer Res 2007;67:2351-2359.
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