Download TAS-116, a Highly Selective Inhibitor of Heat Shock

Published OnlineFirst November 21, 2014; DOI: 10.1158/1535-7163.MCT-14-0219
Molecular
Cancer
Therapeutics
Small Molecule Therapeutics
TAS-116, a Highly Selective Inhibitor of Heat
Shock Protein 90a and b, Demonstrates Potent
Antitumor Activity and Minimal Ocular Toxicity in
Preclinical Models
Shuichi Ohkubo1, Yasuo Kodama1, Hiromi Muraoka1, Hiroko Hitotsumachi2,
Chihoko Yoshimura1, Makoto Kitade1, Akihiro Hashimoto1, Kenjiro Ito1, Akira Gomori1,
Koichi Takahashi1, Yoshihiro Shibata1, Akira Kanoh1, and Kazuhiko Yonekura1
Abstract
The molecular chaperone HSP90 plays a crucial role in cancer
cell growth and survival by stabilizing cancer-related proteins. A
number of HSP90 inhibitors have been developed clinically for
cancer therapy; however, potential off-target and/or HSP90related toxicities have proved problematic. The 4-(1Hpyrazolo[3,4-b]pyridine-1-yl)benzamide TAS-116 is a selective
inhibitor of cytosolic HSP90a and b that does not inhibit HSP90
paralogs such as endoplasmic reticulum GRP94 or mitochondrial TRAP1. Oral administration of TAS-116 led to tumor
shrinkage in human tumor xenograft mouse models accompanied by depletion of multiple HSP90 clients, demonstrating that
the inhibition of HSP90a and b alone was sufficient to exert
antitumor activity in certain tumor models. One of the most
notable HSP90-related adverse events universally observed to
differing degrees in the clinical setting is visual disturbance. A
two-week administration of the isoxazole resorcinol NVPAUY922, an HSP90 inhibitor, caused marked degeneration and
disarrangement of the outer nuclear layer of the retina and
induced photoreceptor cell death in rats. In contrast, TAS-116
did not produce detectable photoreceptor injury in rats, probably due to its lower distribution in retinal tissue. Importantly, in
a rat model, the antitumor activity of TAS-116 was accompanied
by a higher distribution of the compound in subcutaneously
xenografted NCI-H1975 non–small cell lung carcinoma tumors
than in retina. Moreover, TAS-116 showed activity against orthotopically transplanted NCI-H1975 lung tumors. Together, these
data suggest that TAS-116 has a potential to maximize antitumor
activity while minimizing adverse effects such as visual disturbances that are observed with other compounds of this class. Mol
Introduction
for cancer interventions, and many HSP90 inhibitors have been
developed (8).
Though HSP90 inhibitors have been shown in different preclinical models to exert their potent antitumor activities by
destabilizing HSP90 clients (9–13), they have also been shown
to have limited single-agent activity in the clinical setting
(3, 8, 14). One explanation for this discrepancy may be that the
levels of drug exposure reached in patients are insufficient to
effectively inhibit tumor cell HSP90 because of undesirable offtarget and/or HSP90-related adverse events. For example, liver
toxicity was a major limitation in the clinical development of the
first-generation ansamycin derivatives tanespimycin (17-AAG)
and alvespimycin (17-DMAG; ref. 15). It is possible that this
excess liver toxicity is related to the quinone structure of ansamycin (16). The second generation of HSP90 inhibitors are based
on a diverse variety of chemical scaffolds and exhibit less liver
toxicity (11, 13). However, these compounds cause other doselimiting toxicities, some of which are difficult to manage. For
example, neurological toxicities such as syncope and dizziness
were seen in a phase I trial of the purine analogue BIIB021 (17),
although there is no evidence to support this to be target or
purine-scaffold related. In clinical trials of other HSP90 inhibitors,
the most commonly observed HSP90-related adverse events have
been visual disorders such as night blindness, photopsia, blurred
vision, and visual impairment, though the frequency and degree
varied among the compounds (18–21). For example, 43% of
The molecular chaperone heat shock protein 90 (HSP90) is a
highly conserved protein that regulates the activity and stability
of a diverse range of "client" proteins (1, 2). HSP90 clients
include many of the cancer-related proteins necessary for tumor
development, including receptor tyrosine kinases, signal transducers, cell-cycle regulators, and transcriptional factors (1–3).
HSP90 is reported to be specifically overexpressed (4, 5) and to
exist as activated multi-chaperone complexes in cancer cells and
cancer tissues (6, 7); indeed, it has been proposed that cancers
are highly "addicted" to HSP90 for their survival and proliferation (1). HSP90 has therefore emerged as an attractive target
1
Tsukuba Research Center, Taiho Pharmaceutical Co., Ltd., Tsukuba,
Ibaraki, Japan. 2Tokushima Research Center,Taiho Pharmaceutical Co.,
Ltd., Kawauchi-cho, Tokushima, Japan.
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Shuichi Ohkubo, Taiho Pharmaceutical Co., Ltd., 3
Okubo, Tsukuba, Ibaraki 300-2611, Japan. Phone: 81-29-865-4527; Fax: 81-29865-2170; E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-14-0219
2014 American Association for Cancer Research.
Cancer Ther; 14(1); 14–22. 2014 AACR.
14 Mol Cancer Ther; 14(1) January 2015
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Published OnlineFirst November 21, 2014; DOI: 10.1158/1535-7163.MCT-14-0219
Preclinical Characterization of HSP90a/b Inhibitor TAS-116
patients suffered visual disturbances in a phase I trial of NVPAUY922 (21). Though HSP90-related visual disorders, which
have included grade 3 events, are reversible (21), these adverse
events interfere with obtaining sufficient drug exposure because of
drug interruption or discontinuation, or dose reduction.
In this report, we describe our preclinical characterization of
TAS-116, a novel, selective HSP90a and b inhibitor, and show
that, due to limited exposure of nontarget eye tissues and greater
exposure of target tumor tissues to the compound, TAS-116 has a
favorable therapeutic index.
Materials and Methods
Chemical compounds
The HSP90 inhibitors TAS-116 (Fig. 1A), NVP-AUY922,
BIIB021, SNX-2112, SNX-5422, and ganetespib were synthesized
at Taiho Pharmaceutical, Co. Ltd., and 17-AAG and 17-DMAG
were purchased from Biotrend Chemikalien GmbH and LC Laboratories, respectively. Fluorescein isothiocyanate–labeled geldanamycin (geldanamycin-FITC) was purchased from Enzo Life
Sciences International, Inc.
Antibodies
The following antibodies were purchased from Cell Signaling
Technology, Inc.: anti-phospho-HER2 (Tyr1221/1222; #2243);
anti-HER2 (#2165); anti–phospho-HER3 (Tyr1289; #4791);
anti–phospho-p44/42 MAPK (Thr202/Tyr204; #4370); anti-p44/
42 MAPK (#9102); anti–phospho-AKT (Ser473; #4060); anti-AKT
(#4691); anti–phospho-S6 Ribosomal Protein (Ser235/236;
#4858); anti-S6 Ribosomal Protein (#2217); anti-DDR1
(#3917); anti-HSP70 (#4876); and anti-GAPDH (#2118). AntiHER3 (sc-285) was purchased from Santa Cruz Biotechnology, Inc.
Anti-FLT3 (MAB8121) was purchased from R&D Systems Inc.
HSP90 affinity determination
Recombinant human HSP90AA1 (HSP90a), HSP90AB1
(HSP90b), and TRAP1 were purchased from Enzo Life Sciences
International, Inc., and HSP90B1 (GRP94) was purchased from
ATGen Co., Ltd. The interactions of TAS-116 with proteins from
the HSP90 protein family were determined by means of a fluorescence polarization competitive binding assay with geldanamycin-FITC (22, 23). Each compound was incubated with recombinant HSP90a, HSP90b, GRP94, or TRAP1 for 2 hours. Geldanamycin-FITC was added and the mixture was incubated for a
further 5 hours. The concentration of each compound required to
reduce by 50% the amount of recombinant proteins bound to
geldanamycin-FITC (IC50) was determined and Ki values were
calculated from the IC50 values with the Cheng–Prusoff equation
(23, 24).
Western blot analysis
Cells were lysed in lysis buffer [M-PER Mammalian Protein
Extraction Reagent (Thermo Fisher Scientific Inc.) supplemented
with cOmplete, Mini, Protease Inhibitor Cocktail (Roche Applied
Science) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche
Applied Science)]. Proteins were separated by SDS-PAGE and
transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc.). Membranes were blocked with Blocking One or
Blocking One P blocking reagent (Nacalai Tesque, Inc.) and
probed with the appropriate primary antibodies. The membranes
were then incubated with horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology, Inc.), and proteins
were visualized by means of luminol-based enhanced chemiluminescence (Thermo Fisher Scientific Inc.). Luminescent images
were captured with an LAS-3000 imaging system (Fuji Photo Film
Co., Ltd.).
Cell lines and cell culture
All cell lines were purchased from American Type Culture
Collection in 2008 and 2009, and reauthenticated by means of
short tandem repeat–based DNA profiling in 2012. Cells were
cultured in McCoy's 5A medium (HCT116), RPMI 1640 medium
(NCI-N87 and NCI-H1975), or Iscove's modified Dulbecco's
medium (MV-4-11) supplemented with 10% FBS.
Proximity ligation assay
Cells were grown on Lab-Tek II Chambered Coverglass
(Thermo Fisher Scientific Inc.), and then treated with TAS-116
or 17-AAG for 8 hours. The cells were then fixed in 4% paraformaldehyde and blocked in Duolink II solution (Sigma-Aldrich
Co. LLC). The slides were incubated with anti-LRP6 rabbit
(ab134146; Abcam plc) and anti-GRP94 mouse (AM09037PUS; Acris Antibodies, Inc.) antibodies, followed by incubation with
A
C
B
N
TAS-116
N
0
N
0.3
1
3
17-AAG
0.1 0.3 1
(µmol/L)
Untreated
TAS-116, 3 µmol/L
17-AAG, 1 µmol/L
LRP6
N
N
N
DDR1
N
GRP94
HSP70
H2N
O
GAPDH
Figure 1.
Effects of TAS-116 on the levels of HSP90-related proteins and GRP94/LRP6 complex formation in HCT116 cells. A, chemical structure of TAS-116, 3-ethyl-4-[3-(1methylethyl)-4-[4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl]-1H-pyrazolo[3,4-b]pyridin-1-yl]benzamide. B, dose-dependent change of HSP90-related
proteins in HCT116 cells treated with TAS-116 or 17-AAG for 8 hours. Western blotting was performed by using 10 mg of cell lysate. C, in situ proximity ligation assay in
HCT116 cells treated with 3 mmol/L of TAS-116 or 1 mmol/L of 17-AAG for 8 hours. Primary mouse and rabbit antibodies against GRP94 and LRP6 were combined with
secondary proximity ligation assay probes (Sigma-Aldrich Co., LLC.). Interaction events are visible as red dots. Nuclei were stained blue. Scale bar, 20 mm.
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15
Published OnlineFirst November 21, 2014; DOI: 10.1158/1535-7163.MCT-14-0219
Ohkubo et al.
Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity
probes (Sigma-Aldrich Co. LLC). The proximity ligation assay
was performed by using a Duolink detection reagent kit (SigmaAldrich Co. LLC) according to the manufacturer's instructions.
Nuclei were visualized by means of Hoechst 33342 (Nacalai
Tesque, Inc.) staining. Stained cells were imaged by using an LSM
510 confocal microscope with a Plan-Apochromat 20/0.8 objective (Carl Zeiss AG) and the LSM Image Browser software.
In vivo efficacy studies
Cancer cells were subcutaneously implanted into six-week-old
male BALB/c nude mice (Clea Japan, Inc.) or six-week-old male
F344 nude rats (Clea Japan, Inc.) and allowed to grow. Five or six
animals were assigned to each group for each experiment, and
TAS-116 formulated in 0.5% (w/v) hydroxypropyl methylcellulose (Metolose 60SH-50; Shin-Etsu Chemical Co., Ltd.) in H2O
was administered orally every day at 3.6 to 14.0 mg/kg/day.
Tumor volume was calculated with the formula [length (width)2]/2. Statistical significance was calculated by using either
Dunnett or Student t test to assess the difference in tumor volumes
between control (either vehicle or untreated) and TAS-116–treated groups. P < 0.05 was considered statistically significant. For
pharmacodynamic analysis, tumors were harvested at the indicated time points after administration of TAS-116. The excised
tumors were homogenized in lysing matrix D (MP Biomedicals,
LLC) containing lysis buffer, and lysates were prepared.
To establish an orthotopic lung tumor model, NCI-H1975
was engineered to stably express luciferase (H1975-Luc) by
using a lentiviral vector (Life Technologies Corp.). H1975-Luc
cells (1 106 cells/mouse) were mixed with Matrigel (BD Biosciences) and directly transplanted into the left lobe of the eightweek-old BALB/c nude mouse lung. Six weeks after transplantation, TAS-116 at 14.0 mg/kg/day was orally administered five
times a week for four weeks. Mice were intravenously administered D-luciferin potassium salt (Promega Corp.) at a dose of 150
mg/kg, and then dorsal and ventral bioluminescence images were
taken with an IVIS Lumina II Imaging System (Perkin Elmer).
Images were analyzed with the Living Image 3.1 software (Perkin
Elmer). All animal experiments were performed with the approval
of the institutional animal care and use committee of Taiho
Pharmaceutical Co., Ltd. and carried out according to the guidelines for animal experiments of Taiho Pharmaceutical Co., Ltd.
Histopathologic examination of rat eye tissues
TAS-116 was administered orally every day to six-week-old
male Sprague Dawley (SD) rats (Charles River Laboratories Japan,
Inc.) and six-week-old male Long–Evans (LE) rats (Institute for
Animal Reproduction) for two weeks. NVP-AUY922 formulated
in 5% (v/v) dimethyl sulfoxide and 5% (v/v) Tween 20 in saline
was administered intravenously to SD rats three times a week for
two weeks at 10.0 mg/kg/day. Eyes were harvested 24 hours after
the final dose, fixed in Davidson solution for 48 hours, and stored
in 10% (v/v) buffered formalin solution before routine paraffin
embedding and sectioning. Eye sections underwent hematoxylin
and eosin (HE) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; ApopTag Plus Peroxidase
In Situ Apoptosis Detection Kit; Merck Millipore).
Quantitation of plasma, tumor, and retinal tissue exposure to
the test compounds
Test compounds were administered orally or intravenously to
SD, LE, or tumor-bearing F344 nude rats. Plasma, retina, and/or
tumor samples were collected according to the sampling schedule
after dosing. Retina or tumor samples were homogenized in PBS.
Plasma and homogenized samples were analyzed by means of
liquid chromatography with tandem mass spectrometry or highperformance liquid chromatography. The pharmacokinetic parameters of TAS-116 in mice, rats, and dogs were calculated by using
noncompartmental methods (25).
Results
TAS-116 selectively inhibits HSP90a and b
TAS-116 (Fig. 1A) is a novel, small-molecule HSP90 inhibitor
that was discovered during a multiparameter lead optimization
campaign to have high target-selectivity for certain HSP90 proteins (26). In the present study, TAS-116 inhibited geldanamycin–
FITC binding to HSP90 proteins with Ki values of 34.7 nmol/L,
21.3 nmol/L, >50,000 nmol/L, and >50,000 nmol/L for HSP90a,
HSP90b, GRP94, and TRAP1, respectively (Table 1). Other HSP90
inhibitors, including 17-AAG, 17-DMAG, NVP-AUY922, BIIB021,
and SNX-2112, also inhibited GRP94 and TRAP1 in addition
to HSP90a and b, though generally these compounds inhibited
GRP94 and TRAP1 less than they did HSP90a and b. Furthermore,
TAS-116 did not inhibit other ATPases such as HSP70 (IC50,
>200 mmol/L; data not shown) or any of 48 different protein
kinases tested (IC50, all >30 mmol/L; Supplementary Table S1).
The HSP90 selectivity of TAS-116 was further evaluated at the
cellular level. Treatment of HCT116 cells with either 0.1 mmol/L of
17-AAG or 0.3 mmol/L of TAS-116 for 8 hours resulted in reduced
levels of DDR1, which interacts with HSP90a (27), and induction
of HSP70, which is a surrogate marker of cytosolic HSP90 inhibition (3, 28), at comparable levels for both compounds (Fig. 1B),
thereby demonstrating the compounds' cytosolic HSP90 inhibitory effects. GRP94 interacts with low-density lipoprotein receptor-related protein 6 (LRP6) and has a role in chaperoning LRP6
(29). Therefore, LRP6/GRP94 complex formation was evaluated
in HCT116 cells in situ by means of a proximity ligation assay in
which PLA-probe–labeled LRP6 or GRP94 molecules in close
proximity were detected as red dots (30). LRP6/GRP complex
Table 1. Binding affinity of HSP90 inhibitors to proteins in the HSP90 protein family
Compound
TAS-116
17-AAG
17-DMAG
NVP-AUY922
BIIB021
SNX-2112
HSP90a
34.7 8.4
10.0 1.3
5.8 0.5
4.5 0.1
6.1 0.9
11.5 1.8
Binding affinity of HSP90 family proteins (Ki, nmol/L)
HSP90b
GRP94
21.3 3.0
>50,000
6.6 0.3
19.9 4.2
4.8 0.4
12.6 1.0
3.2 0.1
27.0 2.6
5.5 0.3
117.6 23.7
9.0 0.5
720.2 22.1
TRAP1
>50,000
968.4 405.0
110.7 14.7
71.8 4.8
101.9 5.1
533.1 58.0
NOTE: Data are presented as mean SEM (n ¼ 9).
16 Mol Cancer Ther; 14(1) January 2015
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Preclinical Characterization of HSP90a/b Inhibitor TAS-116
Table 2. Pharmacokinetic profiles of TAS-116 in rodent and nonrodent species
Species
n
Dose (mg/kg)
tmax (h)
Cmax (mg/mL)
Mouse
3
3.6
1.0
1.8
3
7.1
2.0
3.1
3
14.0
1.0
6.7
Rat
4
4.0
1.4
1.4
Dog
3
3.0
2.7
0.6
t1/2 (h)
8.2
2.5
4.4
2.2
3.9
AUC0–24 (mgh/mL)
16.4
28.3
60.2
8.4
5.1
F (%)
100
100
100
69.0
73.9
Abbreviations: AUC, area under the concentration-versus-time curve; Cmax, maximum plasma concentration; F, bioavailability; tmax, time of maximum concentration;
t1/2, elimination half-life.
was observed in untreated cells; however, treatment with 1
mmol/L of 17-AAG for 8 hours caused a reduction in the amount
of LRP6/GRP94 complex detected (Fig. 1C) and slightly reduced
the level of LRP6 (Fig. 1B), which is dependent on GRP94 for its
cell-surface expression (29). In contrast, 3 mmol/L of TAS-116 did
not markedly affect the LRP6–GRP94 interaction. However, TAS116 was unable to be used at higher concentrations because
HCT116 cells became detached from the glass slide at concentrations of TAS-116 above 3 mmol/L. Our data suggest that 3 mmol/L
of TAS-116 does not inhibit GRP94, whereas 0.3 mmol/L of TAS116 inhibits HSP90a and b in HCT116 cells.
TAS-116 shows favorable pharmacokinetics
Pharmacokinetic profiling of TAS-116 in rodent and nonrodent
species showed that TAS-116 was orally absorbed and had a
bioavailability of almost 100% in mice, 69.0% in rats, and
73.9% in dogs without special formulation (Table 2). In addition,
the inhibitory activity of TAS-116 on cytochrome P450 (CYP)
enzymes was minimal; only a limited number of isoforms were
inhibited (e.g., IC50, 21.6 mg/mL and 18.5 mg/mL for CYP2C8 and
CYP2C9, respectively; Supplementary Table S2) above the effective concentration of TAS-116 (Cmax of TAS-116 at a dose of
14.0 mg/kg in mice was 6.7 mg/mL; Fig. 2A). Thus, it may be
possible to combine TAS-116 with other anticancer agents without inducing clinically relevant drug–drug interactions.
TAS-116 exhibits potent antitumor activity accompanied by
depletion of HSP90 clients in human tumor xenograft models
The antitumor activity of TAS-116 was evaluated in a HER2expressing NCI-N87 (31) human gastric cancer xenograft mouse
model. The favorable pharmacokinetic profile of TAS-116
was reflected in its dose-dependent antitumor activity; the T/C
(tumor volume of TAS-116–treated mice vs. vehicle-treated mice)
was 47%, 21%, and 9% for doses of 3.6 mg/kg, 7.1 mg/kg, and
14.0 mg/kg, respectively (Fig. 2A). Chronic administration of TAS116 was tolerable, with the average weight loss in mice not
exceeding 10% during the treatment period (Fig. 2B). In the
pharmacodynamic analysis, TAS-116 (14.0 mg/kg) downregulated
HER2, HER3, and AKT protein levels, leading to inhibition of PI3K/
AKT and MAPK/ERK signaling in xenografted tumors (Fig. 2C).
We then determined the antitumor activity of TAS-116 in an
FLT3-ITD–expressing human acute myeloid leukemia (MV-4-11;
ref. 32) xenograft mouse model. Oral administration of TAS-116
at a dosage of 14.0 mg/kg/day for 14 days led to tumor shrinkage
(Fig. 2D), accompanied by depletion of FLT3-ITD (Fig. 2E) in this
model.
TAS-116 shows antitumor activity without inducing eye injury
in rats
In clinical trials of HSP90 inhibitors, visual disturbance has
been a frequently observed adverse event. Indeed, ocular toxicity
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has been seen even in preclinical models (33). It is possible that
ocular toxicity is associated with the extent to which eye tissues are
exposed to the drug (33). Therefore, we evaluated the tissue
distribution profile and ocular toxicity of TAS-116 in rats bearing
subcutaneous human tumors (NCI-H1975). NVP-AUY922 was
used as the reference compound because visual impairment was
frequently observed in a phase I study of this compound (21).
After administration of NVP-AUY922 intravenously or TAS-116
orally, plasma, retina, and tumor samples were collected and the
concentration of each compound in the tissues was determined.
Whereas NVP-AUY922 was distributed more in retina than plasma and was detectable at 24 hours after administration, TAS-116
was distributed less in retina than plasma and was eliminated
from the retina within 24 hours in tumor-bearing nude rats
(Fig. 3A). TAS-116 was also distributed less in retina than in
plasma after intravenous administration (data not shown), indicating the rapid establishment of a distribution equilibrium
between plasma and retina.
In a histopathologic examination of eye tissues to evaluate
the ocular toxicity of the compounds, NVP-AUY922 was
administered intravenously at 10.0 mg/kg/day three times a
week for two weeks and TAS-116 was administered orally at
12.0 mg/kg/day every day in albino SD rats. NVP-AUY922
caused marked degeneration and disarrangement of the outer
nuclear layer of the retina, and the numbers of TUNEL-positive
apoptotic cells increased according to the retinal distribution of
the compound (Fig. 3B). In contrast, administration of TAS-116
resulted in no histologic changes, no abnormalities in the
photoreceptor cell ratio, and no increases in the number of
TUNEL-positive cells in the outer nuclear layer of the retina in
SD rats. Although retinal pigment epithelium has numerous
functions in the maintenance of the integrity and function of
photoreceptors (34), TAS-116 was less distributed in retina
than in plasma and did not cause photoreceptor injury in
pigmented LE rats (data not shown).
TAS-116 showed a much higher distribution in tumor
than in retina in tumor-bearing rats (Fig. 3A). Importantly, a
two-week administration of TAS-116 at a dose of 12.0 mg/kg/
day resulted in antitumor activity accompanied by depletion
of HSP90 client, mutated-EGFR, leading to inhibition of PI3K/
AKT and MAPK/ERK signaling in the NCI-H1975 rat xenograft
model (Fig. 3C and D). Again, TAS-116 did not produce
detectable photoreceptor injury in this model (data not
shown). These data suggested that TAS-116 did not cause ocular
toxicity at the effective dose in rats.
To further evaluate the tissue distribution profiles of other
structurally unrelated HSP90 inhibitors, we also measured
plasma, retinal, and tumoral exposure to 17-DMAG, ganetespib, and SNX-5422 in a subcutaneous xenograft rat model.
Though all the HSP90 inhibitors tested, including NVPAUY922 and TAS-116, were highly distributed and retained in
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Ohkubo et al.
A
B
1,000
30
Control
Control
TAS-116, 3.6 mg/kg
Body weight change (%)
Tumor volume (mm3)
750
TAS-116, 3.6 mg/kg
20
TAS-116, 7.1 mg/kg
TAS-116, 14.0 mg/kg
500
**
T/C = 47%
250
**
T/C = 21%
TAS-116, 7.1 mg/kg
TAS-116, 14.0 mg/kg
10
0
−10
−20
** T/C = 9%
−30
0
5
10
15
Treatment day
C
20
0
5
10
D
20
E
t
TA rol
S11
6
1,250
on
6
Control
p-HER3 (Y1289)
HER3
p-ERK1/2 (T202/Y204)
ERK1/2
p-AKT (S473)
AKT
FLT3-ITD
750
GAPDH
500
250
**
p-RPS6 (S235/236)
RPS6
GAPDH
0
0
5
10
Treatment day
tumor (Fig. 3A and Supplementary Fig. S1), as previously
reported (9, 11, 35, 36), TAS-116 was more rapidly eliminated
from retina (t1/2 ¼ 3.4 hours) than the other HSP90 inhibitors
(t1/2 ¼ 7.1–19.1 hours), thereby demonstrating its lower retinal
distribution profile (Fig. 3A and Supplementary Table S3).
Furthermore, the drug exposure ratio between tumor and retina
of each compound (T/R ratio) was calculated to assess which
compound was distributed more in tumor than in retina; TAS116 showed the highest T/R ratio (T/R ¼ 5.7) from among the
tested HSP90 inhibitors (T/R ¼ 0.7–2.3), indicating its unique
tissue distribution profile (Fig. 3E).
TAS-116 significantly suppresses tumor growth in an
orthotopic lung tumor model expressing EGFR (L858R/T790M)
TAS-116 demonstrated antitumor activity without ocular toxicity in a subcutaneous xenograft model. However, it is possible
that the favorable tissue distribution profile of TAS-116 is only
observed under such subcutaneous conditions. To rule out this
possibility, we evaluated the antitumor activity of TAS-116 by
using an orthotopic human lung cancer model in mice. Luciferasetransduced NCI-H1975 cells harboring EGFR (L858R/T790M;
ref. 37) were directly transplanted into the lungs of mice, and
TAS-116 was administered six weeks after transplantation. Tumor
volume was assessed by means of bioluminescence imaging
(Fig. 4). Although the intensity of luminescence was increased
in the vehicle-treated group, TAS-116 administration resulted in
significantly decreased luminescence. These results suggested that
the tumor growth in the mouse lung was suppressed by treatment
with TAS-116.
18 Mol Cancer Ther; 14(1) January 2015
S-
on
TAS-116, 14.0 mg/kg
1,000
Tumor volume (mm3)
HER2
C
p-HER2 (Y1221/1222)
TA
tro
l
C
15
Treatment day
11
0
Figure 2.
Antitumor activity of TAS-116 in
human tumor xenograft mouse
models. Antitumor activity of TAS-116
in NCI-N87 (A) and MV-4-11 (D)
xenograft models. Athymic nude mice
bearing the indicated tumors
(n ¼ 6 mice per group) were orally
administered TAS-116 at 3.6, 7.1, or
14.0 mg/kg/day. T/C, tumor volume of
TAS-116-treated mice versus vehicletreated mice. Data are presented as
mean SD. , P < 0.01 versus control
[either vehicle-treated (NCI-N87) or
untreated (MV-4-11)]. B, body weight
change of mice corresponding to A.
Effects of TAS-116 treatment on
HSP90 client protein levels in an
NCI-N87 (C) or MV-4-11 (E) mouse
xenograft model. Mice bearing
NCI-N87 tumors were orally
administered TAS-116 at 14.0 mg/kg/
day for 3 days. Mice bearing MV-4-11
tumors were treated with a single oral
dose of 14.0 mg/kg of TAS-116. Mice
were euthanized at 12 hours after the
final dose, and tumors were harvested.
Protein extracts (20 mg) from
tumors were subjected to Western
blot analysis.
15
Discussion
HSP90 inhibitors simultaneously affect multiple cancer-related
proteins, and are therefore potentially useful for treating targeted
therapy–resistant tumors that have arisen from mutations within
the target itself or from activation of alternative signaling pathways. For example, NVP-AUY922 is active against ALK-TKI– or
EGFR-TKI–resistant non–small cell lung carcinoma (8, 38). However, because HSP90 also plays a role in normal cells, it is essential
to balance efficacy and toxicity to maximize the antitumor potential of HSP90 inhibitors. Here, we characterize a novel HSP90
inhibitor that showed superior druggability with respect to target
selectivity and its pharmacologic, pharmacokinetic, and safety
profiles.
TAS-116 represents one of the most selective HSP90a and b
inhibitors developed to date; it shows greater specific binding to
HSP90a and b than to the highly homologous HSP90 family
members GRP94 and TRAP1. The relative importance of the four
HSP90 paralogs with respect to cancer treatment remains poorly
understood. Although inhibition or downregulation of GRP94
inhibits growth and induces apoptosis in human multiple myeloma and HER2-expressing cell lines (39, 40), the role of GRP94
in liver tumorigenesis remains controversial, with recent studies
indicating the potential for GRP94 to act as both a tumor suppressor and an oncogene (41, 42). Inhibition of TRAP1 by a
mitochondria-directed variant of 17-AAG leads to specific apoptosis in cancer cells (43); however, TRAP1 may also play, in a
context-dependent manner, a tumor suppressor or an oncogene
role (44). Our present finding that the highly selective HSP90a
Molecular Cancer Therapeutics
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Published OnlineFirst November 21, 2014; DOI: 10.1158/1535-7163.MCT-14-0219
Preclinical Characterization of HSP90a/b Inhibitor TAS-116
Concentration of NVP-AUY922 (µmol/L)
TAS-116 (po)
14
Concentration of TAS-116 (µmol/L)
A
Plasma
12
Retina
10
Tumor
8
6
4
2
0
0
6
12
18
24
NVP-AUY922 (iv)
14
Plasma
12
Retina
10
Tumor
8
6
4
2
0
0
Time after administration (h)
B
6
12
18
24
Time after administration (h)
TAS-116
(12.0 mg/kg, po, qdx14)
Control
NVP-AUY922
(10.0 mg/kg, iv, 3x/w)
HE
ONL
INL
GCL
ONL
ONL
ONL
TUNEL
ONL
INL
GCL
C
D
# 1
2
3
Control
TAS-116, 12.0 mg/kg
TAS-116
(12.0 mg/kg)
1
2
3
EGFR (L858R/T790M)
p-ERK1/2 (T202/Y204)
ERK1/2
p-AKT (S473)
AKT
p-RPS6 (S235/236)
RPS6
Tumor volume (mm3)
Control
E
10,000
TAS-116
8,000
SNX-5422
6,000
17-DMAG
4,000
Ganetespib
**
2,000
NVP-AUY922
GAPDH
0
0
5
10
Treatment day
15
0.1
1.0
10
AUC0–24, tumor / AUC0–24, retina
Figure 3.
Comparative pharmacokinetic and retinal toxicity analysis of TAS-116 and NVP-AUY922. A, biodistribution of TAS-116 or NVP-AUY922 in athymic nude rats with
established NCI-H1975 xenografts after oral or intravenous administration of TAS-116 at 12.0 mg/kg or NVP-AUY922 at 20.0 mg/kg, respectively. Data are
presented as mean SD (n ¼ 3). po, oral administration; iv, intravenous administration. B, effects of TAS-116 or NVP-AUY922 on retinal morphology and
photoreceptor cell death in SD rats. TAS-116 was administered orally every day for two weeks. NVP-AUY922 was administered intravenously three times a week for
two weeks. Eye sections were subjected to HE staining and TUNEL. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; po qd, daily oral
administration; w, week. Scale bars, 50 mm. Effect on HSP90 clients (C) and antitumor activity of TAS-116 (D) in an NCI-H1975 rat xenograft model. For
pharmacodynamic analysis, rats with established NCI-H1975 xenografts were treated with an oral dose of 12.0 mg/kg of TAS-116. Tumors (n ¼ 3 per group) were
harvested at 8 hours after administration. The levels of the HSP90 clients were evaluated by means of Western blotting. To evaluate the antitumor activity, NCI-H1975
tumor–bearing rats (n ¼ 5 rats per group) were orally treated with TAS-116 at 12.0 mg/kg daily for two weeks. , P < 0.01 versus untreated control. E, comparison
of the drug exposure ratio between tumor and retina (AUC0–24, tumor/AUC0–24, retina) of HSP90 inhibitors in an NCI-H1975 rat xenograft model. HSP90 inhibitors
were administered in tumor-bearing rats with the routes and doses described in Supplementary Table S3 and then subjected to pharmacokinetic analysis.
www.aacrjournals.org
Mol Cancer Ther; 14(1) January 2015
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19
Published OnlineFirst November 21, 2014; DOI: 10.1158/1535-7163.MCT-14-0219
Ohkubo et al.
A
D
V
D
V
D
V
D
V
D
Control
V
10.0
9.0
TAS-116, 14.0 mg/kg
8.0
Day -5
V
Day 8
D
V
Day 15
D
V
Day 22
D
V
7.0 × 108
Day 29
D
V
D
6.0
5.0
4.0
B
Total photon (V+D, photons/s)
1.E+12
Control
TAS-116, 14.0 mg/kg
1.E+11
**
1.E+10
1.E+09
1.E+08
−42 −35 −28 −21 −14 −7
0
7
14 21 28
Days after start of administration
Figure 4.
Effects of TAS-116 in a lung cancer mouse model based on orthotopically xenografted luciferase-transduced NCI-H1975 cells. A, representative photographs of
photon emissions from individual mice with an orthotopic lung tumor with or without TAS-116 administration. Color gradation scale indicates the number of
live tumor cells (purple, low bioluminescence; red, high bioluminescence). V, ventral image; D, dorsal image; ROI, region of interest. B, emitted bioluminescence
(photons/s) at the indicated time points (n ¼ 8 mice per group). Data are presented as mean SD. , P < 0.01 versus vehicle-treated control.
and b inhibitor TAS-116 has strong antitumor activity against
several xenografted tumors indicates that inhibition of the
HSP90a and b members of the HSP90 family is sufficient to
exert antitumor activity.
The impact of paralog selectivity on the therapeutic index
and toxic effects of HSP90 inhibitors is yet to be sufficiently
explored (3). Recently, it was discovered that GRP94, a critical
chaperone for the Wnt coreceptor LRP6, has a functional role in
gut homeostasis via regulation of the canonical Wnt signaling
pathway (29). Conditional deletion of grp94 results in rapid
weight loss, diarrhea, and hematochezia in mice that are characterized by a gut-intrinsic defect in the proliferation of intestinal
crypts, as well as compromised nuclear b-catenin translocation,
loss of crypt–villus structure, and impaired barrier function (29).
It however remains unclear whether pharmacologic inhibition of
GRP94 would mimic such effect. Gastrointestinal toxicity is a
major clinical adverse event associated with HSP90 inhibitors
(17–19, 21, 45) that is considered an on-target effect of HSP90
inhibition. However, because TAS-116 did not inhibit the interaction between GRP94 and LRP6 in the present study, it is
conceivable that a selective inhibitor such as TAS-116 may not
20 Mol Cancer Ther; 14(1) January 2015
cause GRP94-related gastrointestinal toxicity. This however
remains to be validated.
One approach to balancing the efficacy and toxicity of
antitumor agents is optimization of the dosing schedule. In
this regard, oral rather than intravenous administration is
preferable as it provides greater dose and schedule flexibility,
and TAS-116 has been shown to have high oral availability in
several species without the need for special formulation or
prodrug development.
Sustained tumoral exposure with minimal exposure of other
organs is another approach to achieve a maximal therapeutic
window for antitumor agents. TAS-116 addresses the issues that
have hampered the clinical development of HSP90 inhibitors
because it shows a favorable tissue distribution profile. One of
the most notable HSP90-related adverse events universally
observed but to differing degrees in the clinic is visual disturbance (18–21, 45–48). The observed symptoms include night
blindness, photopsia, blurred vision, and visual impairment,
which are likely associated with retinal dysfunction (20, 21).
Two independent reports of rodent models suggest that HSP90
plays a critical role in retinal function and that sustained HSP90
Molecular Cancer Therapeutics
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Published OnlineFirst November 21, 2014; DOI: 10.1158/1535-7163.MCT-14-0219
Preclinical Characterization of HSP90a/b Inhibitor TAS-116
inhibition might adversely affect visual function (34, 49). Therefore, ocular effects may be minimized by reducing retinal exposure. In the present study, TAS-116 was distributed less in retina
than in plasma in rats; consequently, TAS-116 did not produce
any detectable photoreceptor injury. It has been proposed that
the retina/plasma drug-exposure ratio and retinal elimination
profile might be useful indicators of the ocular toxic potential of
HSP90 inhibitors (33). Indeed, our results are consistent with
previous results showing that ganetespib does not cause the
same degree of visual symptoms and is eliminated from retina
more rapidly than other HSP90 inhibitors (33). However, there
is the possibility that the lower retinal distribution of some
HSP90 inhibitors is merely correlated with lower tissue penetration of the compounds. If so, such compounds weaken
the risk of ocular toxicities; however, this is at the cost of less
antitumor potential. Importantly, in addition to the lower
retinal distribution, TAS-116 showed a much higher distribution in tumor and antitumor activity in an NCI-H1975 subcutaneous xenograft rat model. To assess the risk-to-benefit
ratio of HSP90 inhibitors, we calculated the tumor/retina (T/
R) drug-exposure ratio for several HSP90 inhibitors in this
xenograft setting. Except for TAS-116, all the tested compounds
showed comparable drug exposure between tumor and retina
(T/R ¼ 0.9–2.3). However, TAS-116 demonstrated a higher T/R
ratio (T/R ¼ 5.7), indicating that it has a unique, favorable tissue
distribution profile. Though the molecular mechanisms by
which TAS-116 achieves this higher T/R exposure ratio remain
to be elucidated, we expect TAS-116 to show a higher therapeutic
window in the clinic than the other HSP90 inhibitors with
respect to ocular toxicity. The drug T/R exposure ratio may also
be useful in future preclinical screenings of candidate HSP90
inhibitor compounds that show an improved therapeutic index.
In conclusion, TAS-116 is an orally available HSP90 inhibitor
that selectively binds to HSP90a and b. TAS-116 showed antitumor activity without detectable ocular toxicities in a rodent
model, probably due to its favorable tissue distribution. Likewise, TAS-116 did not inhibit CYP450 within the effective
concentration range, indicating a low drug–drug interaction
potential that might allow combination administration with
other drugs. These tractable yet specific properties of TAS-116
support the selection of this compound as a candidate for
clinical evaluation as an antitumor agent characterized by maximal antitumor activity and minimal HSP90-related toxicities
such as ocular toxicity.
Disclosure of Potential Conflicts of Interest
S. Ohkubo, C. Yoshimura, and M. Kitade have ownership interest in a patent,
WO2011004610 A1. No potential conflicts of interest were disclosed by the
other authors.
Authors' Contributions
Conception and design: S. Ohkubo, Y. Kodama, C. Yoshimura
Development of methodology: Y. Kodama, H. Muraoka, H. Hitotsumachi,
A. Gomori, K. Takahashi, Y. Shibata
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): S. Ohkubo, Y. Kodama, H. Muraoka, H. Hitotsumachi,
C. Yoshimura, A. Hashimoto, K. Ito, A. Gomori, K. Takahashi, Y. Shibata,
A. Kanoh
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): S. Ohkubo, Y. Kodama, H. Muraoka, H. Hitotsumachi, C. Yoshimura, A. Hashimoto, K. Ito, A. Gomori, K. Takahashi, Y. Shibata
Writing, review, and/or revision of the manuscript: S. Ohkubo, Y. Kodama,
C. Yoshimura
Study supervision: S. Ohkubo, K. Yonekura
Other (chemical design and synthesis): M. Kitade
Acknowledgments
The authors thank Drs. Teruhiro Utsugi and Fumio Morita for their insightful
discussions, and Takamasa Suzuki for his technical assistance.
Grant Support
All research work was funded by Taiho Pharmaceutical Co., Ltd.
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 March 13, 2014; revised October 13, 2014; accepted November 9,
2014; published OnlineFirst November 21, 2014.
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Molecular Cancer Therapeutics
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Published OnlineFirst November 21, 2014; DOI: 10.1158/1535-7163.MCT-14-0219
TAS-116, a Highly Selective Inhibitor of Heat Shock Protein 90α and
β, Demonstrates Potent Antitumor Activity and Minimal Ocular
Toxicity in Preclinical Models
Shuichi Ohkubo, Yasuo Kodama, Hiromi Muraoka, et al.
Mol Cancer Ther 2015;14:14-22. Published OnlineFirst November 21, 2014.
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