Ganetespib (STA-9090), a Non-Geldanamycin HSP90 Inhibitor, has Potent Antitumor Activity in In Vitro and In Vivo Models of Non-Small Cell Lung Cancer
Takeshi Shimamura1,2, Samanthi A. Perera1,2,3, Kevin P. Foley4, Jim Sang4, Scott J. Rodig5,6, Takayo Inoue4, Liang Chen1,2,3, Danan Li1,2,3, Julian Carretero1,2,
Yu-Chen Li1, Papiya Sinha5, Christopher D. Carey5, Christa L. Borgman1, John-Paul Jimenez4, Matthew Meyerson1,6,7, Weiwen Ying4, James Barsoum4,
Kwok-Kin Wong1,2,3, Geoffrey I. Shapiro1,2
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 2Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 3Ludwig Center at Dana-Farber/Harvard Cancer Center, Boston Massachusetts 4Synta Pharmaceuticals Corp., Lexington, Massachusetts 5Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 6Department of Pathology, Harvard Medical School, Boston, Massachusetts 7The Broad Institute of MIT and Harvard, Cambridge, Massachusetts Present Addresses: TS: Department of Molecular Pharmacology and Therapeutics, Oncology Institute, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL; SAP: Merck & Co., Boston, MA; KF: GlaxoSmithKline, Collegeville, PA; LC: National Institute of Biologic Sciences, Beijing, China; DL: Pfizer Pharmaceuticals, La Jolla, CA; JC: Department of Physiology, Faculty of Medicine and Odontology, University of Valencia, Valencia, Spain; CLB: University of Massachusetts School of Medicine, Worcester, MA, JB: Theracrine, Inc., Cambridge, MA Corresponding Authors: Geoffrey I. Shapiro, M.D., Ph.D., Early Drug Development Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Mayer 446, 450 Brookline Ave., Boston, MA 02215. Phone: 617-632-4942; Fax: 617-632-1977; E-mail: [email protected] Kwok-Kin Wong, M.D., Ph.D., Lowe Center for Thoracic Oncology, Department of Medical Oncology, Dana-Farber Cancer Institute, Mayer 413, 450 Brookline Ave., Boston, MA 02215. Phone: 617-632-6084; Fax: 617-632-7839; E-mail: [email protected] Author Disclosures: K.P.F., J.S., T.I., J.-P.J., W.Y., J.B.: Employment, Synta Pharmaceuticals; J.B.: Stock ownership, Synta Pharmaceuticals; K.K.W., G.I.S.: Sponsored Research Support, Synta Pharamceuticals; G.I.S.: Advisory Board, Synta Pharmaceuticals (uncompensated) Short Title: HSP90 inhibitor ganetespib in NSCLC
Key Words: HSP90, ERBB Receptor Tyrosine Kinases, NSCLC, Non-Geldanamycin
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TRANSLATIONAL RELEVANCE
Several subsets of NSCLC are addicted to oncogenic kinases that are dependent on the HSP90
chaperone for conformational stability, stimulating evaluation of HSP90 inhibitors in this
disease. The index geldanamycin compounds have biochemical and toxicity limitations that
have prompted the development of non-geldanamycin compounds, including ganetespib (STA-
9090), a resorcinol-containing triazolone. In in vitro assays and genotypically-defined NSCLC
cell lines and xenografts, ganetespib is more potent and efficacious than 17-allylamino-17-
demethoxygeldanamycin (17-AAG). Although favorable intratumoral pharmacokinetic
parameters suggest that once-weekly dosing may be adequate, several clients, including mutant
EGFR, are suppressed only transiently following ganetespib exposure, so that superior anti-
tumor activity was observed with more frequent consecutive-day dosing. Ganetespib was active
in a mutant ERBB2 YVMA-driven mouse lung adenocarcinoma model with expected
pharmacodynamic effects. These results justify the continued development of ganetespib in
NSCLC, including EGFR and ERBB2-mutant tumors, with assessment of schedules employing
greater than once-weekly drug administration.
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ABSTRACT
Purpose: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat
shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models.
Experimental Design: The activity of ganetespib was compared to that of the geldnamaycin 17-
AAG in biochemical assays, cell lines and xenografts, and evaluated in an ERBB2 YVMA-
driven mouse lung adenocarcinoma model.
Results: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and
disrupted the association of HSP90 with its co-chaperone, p23, more potently than 17-AAG. In
genomically-defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases,
extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis
with IC50 values ranging 2–30 nM, substantially lower than those required for 17-AAG (20–
3,500 nM). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B
cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing
NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma
and normal tissues but was maintained in tumor with t1/2 58.3 hours, supporting once-weekly
dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-
AAG. However, after a single dose, re-expression of mutant EGFR occurred by 72 hours,
correlating with reversal of anti-proliferative and pro-apoptotic effects. Consecutive day dosing
resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects.
Ganetespib also demonstrated activity against mouse lung adenocarcinomas driven by oncogenic
ERBB2 YVMA.
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Conclusions: Ganetespib has greater potency than 17-AAG and potential efficacy against
several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.
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INTRODUCTION
Heat shock protein 90 (HSP90) is an ATPase-dependent molecular chaperone ubiquitously
expressed in eukaryotic cells (1). HSP90 is essential for the post-translational conformational
maturation and stability of client proteins, including protein kinases, steroid receptors and
transcription factors, many of which are important for the proliferation and survival of cancer
cells (2, 3). In contrast to normal tissues, in which HSP90 is found in a latent, uncomplexed
state, tumor cells contain an abundance of catalytically active HSP90 found in multichaperone
complexes, considered important for their survival in a hypoxic, nutrient-deprived and acidotic
microenvironment, and for the maintenance of overexpressed or mutant kinases to which they
are addicted (4, 5). Relevant to non-small cell lung cancer (NSCLC) (6), where high HSP90
expression correlates with poor survival (7), mutant EGFR (8, 9), ERBB2 (10), MET (11),
mutant B-RAF (12) and the EML4-ALK translocation product (13, 14) are all HSP90-dependent
proteins, degradation of which leads to loss of tumor cell viability in the corresponding
adenocarcinoma subset.
Most HSP90 inhibitors under development target the ATPase activity at the N-terminus (15).
The most characterized agents comprise the geldanamycin class, including the benzoquinone
ansamycin HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG;
tanespimycin) (16). Relatively poor physiochemical properties have prompted its modification,
resulting in water soluble derivatives, including 17-dimethylaminoethylamino-17-
demethoxygeldanamcyin (17-DMAG; alvespimycin) (17), and 17-allylamino-17-
demethoxygeldanamycin hydroquinone hydrochloride (IPI-504; retaspimycin hydrochloride)
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(18), all of which have demonstrated activity in a broad range of preclinical models as well as in
phase 1 and 2 studies, particularly in ERBB2 (HER2)-positive breast cancer (19), acute
myelogenous leukemia (20) and EML4-ALK-dependent NSCLC (21). Nonetheless, these agents
are P-glycoprotein substrates (22), may require NAD(P)H:quinone oxidoreductase-1 (NQO1)-
mediated reduction to a more active hydroquinone metabolite (23), and have caused
gastrointestinal and hepatic toxicities in the clinical setting (15). These limitations have
prompted the development of non-geldanamycin inhibitors of HSP90.
Ganetespib (STA-9090) is a non-geldanamycin resorcinol-containing triazolone compound that
binds to the ATP-binding domain at the N-terminus of HSP90 and is currently in phase 1 and 2
clinical trials in both solid tumors and hematologic malignancies (24, 25). Preclinically,
ganetespib and its derivatives have demonstrated activity with low nanomolar potency against
KIT-dependent mast cell tumors (26), MET-dependent osteosarcoma cell lines (27), Wilms
tumor 1 (WT1)-dependent myeloid leukemias (28) and hematologic malignant cells dependent
on JAK/STAT signaling (29). Here, we have investigated the preclinical pharmacokinetics,
pharmacodynamics and activity of ganetespib in NSCLC cells in comparison to 17-AAG.
Ganetespib demonstrates efficacy in a variety of cell line, xenograft and genetically-engineered
mouse models, including those driven by activated KRAS, mutant EGFR and mutant ERBB2.
Although ganetespib displays prolonged intratumoral half-life, frequent dosing schedules are
required to effectively suppress a subset of client proteins, including mutant EGFR, justifying the
current development plan of a variety of treatment schedules.
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MATERIALS AND METHODS
Cell lines and drug treatments
NSCLC cell lines were obtained from the American Type Culture Collection (ATCC). H3255
and DFCI-LU011 cells were provided by Drs. Bruce Johnson and Pasi Jänne (Dana-Farber
Cancer Institute, Boston, MA). PC9 was a gift from Dr. Takashi Owa (Eisai Co., Ltd., Tsukuba,
Japan). Cell lines were subjected to DNA profiling annually (Short Tandem Repeat Analysis) at
the Dana-Farber Cancer Institute Molecular Pathology Core to confirm their authenticity (30).
All cells were maintained in ATCC-specified growth medium. Ba/F3 cells stably expressing
mutant EGFR or ERBB2 were established as previously described (31-33). Pooled stable cell
lines transformed to IL-3 independence were used for drug sensitivity experiments. Ganetespib
was provided by Synta Pharmaceuticals (Lexington, MA) and both ganetespib and 17-
allylamino-17-demethoxygeldanamycin (17-AAG, LC Labs) were prepared as stock solutions in
DMSO.
Cell proliferation assay
Cell proliferation assays were performed using the CCK-8 colorimetric assay (Dojindo) in at
least duplicate samples according to the manufacturer's specifications. IC50 values were
calculated using Kaleidagraph or Graphpad Prism.
Western blots
Whole-cell lysates were prepared as previously described (9). Protein concentrations were
determined and equivalent amounts (20 μg) were subjected to SDS-PAGE on 4-12% bis-tris
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gradient gels (Invitrogen). The HSP27 antibody was from Enzo Life Sciences. p23 and HSP90α
antibodies were from StressMarq. All other antibodies were from Cell Signaling Technology.
Bands were quantified using ImageJ software (NIH).
HSP90 binding assays
Exponentially growing cells were processed in lysis buffer (20 mM HEPES, pH 7.4, 1 mM
EDTA, 5 mM MgCl2, 100 mM KCl) and incubated with increasing concentrations of 17-AAG or
ganetespib for 30 min at 4°C, and incubated with biotin-GM linked to Dynabeads MyOne
Streptavidin T1 magnetic beads (Invitrogen) for 1 h at 4°C. Beads were washed three times in
lysis buffer and heated for 5 min at 95°C in SDS–PAGE sample buffer (Invitrogen). Samples
were resolved on 4-12% Bis-Tris gradient gel and Western blots were performed using an anti-
HSP90 antibody.
Immunoprecipitation
500 μg of whole cell lysate was immunoprecipitated with 2 μg of mouse anti-p23 monoclonal
antibody (clone JJ6, StressMarq) conjugated with protein A Dynabeads (Invitrogen). Proteins
bound to p23 were resolved on 4-12% bis-tris gradient gels and Western blot was performed
with an anti-HSP90 antibody.
Establishment and treatment of xenografts
Female 7–8-week-old C.B-17 SCID mice (Charles River Laboratories) were maintained under
pathogen-free conditions. All procedures were approved by the Synta Pharmaceutical
Institutional Animal Care and Use Committee. NCI-H1975 or HCC827 cells were cultured as
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above and 0.5 – 1×107 cells were mixed with 50% RPMI 1640/50% Matrigel (BD Biosciences)
and subcutaneously injected into the flanks of SCID mice. For efficacy studies, animals with
100-200 mm3 tumors were then randomized into treatments groups of eight. Tumor volumes (V)
were calculated by the equation V = 0.5236×L×W×T (Length, width, and thickness). Animals
were treated by intravenous bolus tail vein injection at 10 ml/kg with ganetespib formulated in
10/18 DRD (10% DMSO, 18% Cremophor RH 40, 3.6% dextrose and 68.4% water). As a
measurement of in vivo efficacy, the relative size of treated and control tumors [(%T/C) value]
was determined from the change in average tumor volumes of each drug-treated group relative to
the vehicle-treated group, or itself in the case of tumor regression. Body weights were monitored
daily. For biomarker studies, mice bearing NCI-H1975 xenografts were treated with either a
single dose of vehicle or ganetespib, or with 5 daily doses of vehicle or ganetespib, in groups of
3 or 8, and harvested at various time points. Tumors were excised and flash frozen in liquid
nitrogen for preparation of protein lysates or fixed in 10% neutral buffered formalin for
immunohistochemistry.
Pharmacokinetic Analysis
Female 7–8-week-old C.B-17 SCID mice bearing NCI-H1975 xenografts received a single
intravenous (i.v.) dose slightly below the highest non-severely toxic dose (HNSTD, 150 mg/kg).
At time points indicated, mice (n = 3/time point) were sacrificed and plasma and tissues (tumor,
liver, and lung) were harvested. Concentrations of ganetespib in plasma and tissues were
determined by isocratic reversed-phase high-performance liquid chromatography with
electrospray ionization mass spectrometric (HPLC/MS-MS) detection.
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Xenograft immunohistochemistry and image analysis. For Cabenda immunohistochemistry
[EGFR, CD31, DiOC7(3), TUNEL, pimonidazole and BrdUrd], NCI-H1975 tumor xenograft-
implanted SCID mice were treated with 125 mg/kg ganetespib for 6-72 h. At the end of the
experiment mice were administered BrdUrd and pimonidazole to label S phase cells and hypoxic
tumor regions and then 5 min prior to excision mice were administered DiOC7(3) to demarcate
perfused vessels. Following tumor excision, cryosections were cut and sequentially
immunostained to detect markers of tumor vasculature (CD31), proliferation (BrdUrd), apoptosis
(TUNEL), as well as EGFR expression (see Supplementary Materials and Methods for details).
Overall BrdUrd positive staining and average EGFR, TUNEL or pimonidazole intensity was
calculated from images of entire tumor sections following removal of necrotic regions and tissue
artifacts (folds, tears, debris etc).
Additional immunostaining on xenografts harvested from mice treated with vehicle or ganetespib
at 150 mg/kg as a single dose or 25 mg/kg daily x 5 was performed as previously described (34);
see Supplementary Materials and Methods for details). Rabbit anti-EGFR L858R (1:100
dilution, clone 43B2, Cell Signaling Technology), rabbit anti-S6 ribosomal protein (1:100
dilution, clone 5G10, Cell Signaling Technology), rabbit anti-phospho-S6 ribosomal protein
ser235/236 (1:50 dilution, clone D57.2.2E, Cell Signaling Technology), mouse anti-HSP27
(1:1000 dilution, clone G31, Cell Signaling Technolgoy), rabbit anti-HSP70 (1:2500 dilution,
clone K-20, Santa Cruz Biotechnology), and mouse anti-Ki67 (clone MIB1, DAKO) antibodies
were applied to individual slides in DAKO background-reducing diluent for 1 hour. Slides were
washed in 50-mM Tris-Cl, pH 7.4, and detected with the species-appropriate Envision+ kit
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(DAKO) as per manufacturer’s instructions. After further washing, immunoperoxidase staining
was developed using a DAB chromogen (DAKO) and counterstained with hematoxylin.
Stained slides were scanned at 200X magnification using an Aperio ScanScope XT workstation
(Aperio Technology, Inc.). Images were then visualized and digitally annotated (as regions of
interest, ROIs) using ImageScope software (version 10.0.35.1800, Aperio Technology). A
pathologist (S.J.R.) verified that areas of viable tumor were included and dead/necrotic tumor
excluded from the analysis. ROIs were analyzed using a standard analysis algorithm to
quantitate the proportion of area positive for staining (color deconvolution v9.0, Aperio
Technology), expressed as the percentage pixels within the ROI that are positive for staining.
Staining of samples from ganetespib-treated mice was compared to that in the corresponding
vehicle controls to derive the percent change in immunohistochemical staining for each time
point after single and multiple-dose administration.
Generation of the CCSP-rtTA/Tet-op- hHER2YVMA mouse cohorts, drug treatment, MRI
scanning, tumor volume measurement, tissue histology and immunohistochemical analysis.
Previously characterized bitransgenic mice with lung adenocarcinomas driven by ERBB2
YVMA were exposed to a doxycycline-containing diet for 6 to 10 weeks, and subjected to
magnetic resonance imaging (MRI) to document tumor burden (35). Littermates were
subsequently left untreated or treated with vehicle or ganetespib, formulated in DRD, and
administered by i.v. injection at 25 mg/kg/daily. Mice underwent serial MRI imaging to assess
reduction in tumor volume and were subsequently sacrificed for further histologic and
biochemical studies. After Dana-Farber Cancer Institute Animal Care and Use Committee
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approval, all mice were housed in a pathogen-free environment at the Harvard School of Public
Health and were handled in accordance with Good Animal Practice as defined by the Office of
Laboratory Animal Welfare. MRI scanning, image acquisition in coronal and axial planes, and
assessment of tumor volume have been described previously (36, 37). H&E and
immunohistochemical staining were performed on formalin-fixed paraffin sections using
standard procedures (9). HER2 (A0485, DAKO), HSP27 (Cell Signaling), and phospho-S6 (Cell
Signaling) were used. For immunohistochemical quantification, 100 to 200 cells were scored as
0, 1+, and 2+ over each of 2 to 4 high-power fields to determine the average percent strongly
positive cells.
Statistical Analyses
For efficacy analyses, due to a non-normal distribution in the tumor volume datasets, statistical
analyses of significance were performed using a Kruskal-Wallis one-way analysis of variance
(ANOVA) on ranks, followed by the Tukey test multiple comparison procedure. Otherwise,
analyses were performed using the two-tailed, unpaired Student's t test. In both cases, P values <
0.05 were considered statistically significant.
RESULTS
Ganetespib shows greater affinity for HSP90 and induces HSP90-p23 dissociation more
readily than 17-AAG.
We first compared the binding affinities of ganetespib and 17-AAG to HSP90 in competitive
binding assays using biotinylated-geldanamycin (biotin-GM) (Fig. 1A). NCI-1975 NSCLC
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(EGFRL858R/T790M) cell lysates were used as a source of HSP90 and were incubated with
either compound. Ganetespib inhibited the binding of HSP90 to biotin-GM at a concentration as
low as 0.11 μmol/L, whereas 1 μmol/L of 17-AAG was required to inhibit binding to the same
degree, suggesting greater affinity of ganetespib for HSP90.
HSP90 binds to co-chaperones, including p23, in an ATP-dependent manner and this assembly
of the catalytically active complex is a prerequisite for efficient chaperone function (38). To
further characterize the in vitro activity of ganetespib in comparison to 17-AAG, we assessed the
ability of these compounds to disrupt catalytically active HSP90-p23 complexes. Lysates from
NCI-H1975 NSCLC cells were used for immunoprecipitation of p23 in the absence or presence
of ganetespib or 17-AAG followed by Western blotting for HSP90. Both compounds resulted in
a concentration-dependent decrease in the amount of HSP90 in complex with p23, with
ganetespib requiring lower concentration to disrupt complex formation (Fig. 1B). Taken
together, these experiments confirm the ability of ganetespib to bind and inhibit HSP90 and
indicate biochemical superiority over 17-AAG.
Ganetespib destabilizes HSP90 client proteins in NSCLC cells with greater potency than
17-AAG.
We next examined the cellular effects of ganetespib, and its ability to deplete critical client
proteins from NSCLC cells in comparison to 17-AAG. In both NCI-H1975 and HCC827 (EGFR
delE746-A750) cells (Fig. 1C and Supplementary Fig. S1), exposure to ganetespib induced client
protein depletion at lower concentration than 17-AAG. For example, both mutant EGFR and
MET were degraded following exposure to 40 nmol/L of ganetespib, whereas concentrations >
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120 and 370 nmol/L of 17-AAG were required to achieve similar levels of depletion of EGFR
and MET, respectively. Treatment of NCI-H1975 or HCC827 cells with 120 nmol/L ganetespib
resulted in complete depletion of IGF-IR, whereas 1,100 nmol/L of 17-AAG was required for a
similar degree of degradation. As expected, both drugs also extinguished downstream signaling
of the PI3K/mTOR and RAF/MEK/ERK pathways, with a lower concentration of ganetespib
required to achieve decreased expression of phospho-S6 and phospho-ERK (Fig. 1C and
Supplementary Fig. S1). Furthermore, depletion of mutant EGFR in HCC827 cells by
ganetespib resulted in the upregulation of BimEL and its subsequent cleavage into the
proapoptotic subtypes BimL and BimS (Fig. 1D). Induction of Bim is required for EGFR
tyrosine kinase inhibitor (TKI)-induced apoptosis (39), indicating that cell death pathways
mediated by TKIs or HSP90 inhibition in EGFR mutant NSCLC cells share common
downstream effectors.
Ganetespib treatment of NSCLC cells also resulted in the depletion of other receptor tyrosine
kinases more readily than 17-AAG, including the PDGFα receptor overexpressed in NCI-H1703
cells, as well as c-RET in HCC1883 cells and ERBB4 in NCI-H522 cells (Supplementary Fig.
S2).
The comparative efficiency of client depletion by ganetespib and 17-AAG translates to the
inhibition of cell proliferation in a panel of 24 NSCLC cell lines with defined genetic
backgrounds (Supplementary Table S1). Ganetespib inhibited proliferation of these cell lines
with IC50 values ranging 2-30 nmol/L (median IC50 = 6.5 nmol/L). In contrast, IC50 values for
17-AAG ranged from 20 -3,500 nmol/L (median IC50 = 30.5 nmol/L). The improved potency of
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ganetespib occurred across genotypes, including EGFR/ERBB2 mutant (n=11), EGFR wild-type
(n=15), KRAS mutant (n=6), and KRAS wild-type (n=18) (Supplementary Table S2), with mean
IC50 values 5–7-fold lower for ganetespib. Finally, we also examined the relative
antiproliferative effects of ganetespib and 17-AAG in Ba/F3 cells ectopically expressing various
mutant EGFRs that render these cells IL-3-independent. In this isogenic system, ganetespib was
also substantially more potent (Supplementary Table S3).
Ganetespib accumulates in tumors relative to normal tissues and displays greater in vivo
efficacy than 17-AAG without increased toxicity
The pharmacokinetic parameters of ganetespib were evaluated in vivo using mice bearing NCI-
H1975 xenografts. Ganetespib was administered as a single dose intravenously at 125 mg/kg
(slightly below the highest non-severely toxic dose (HNSTD) of 150 mg/kg), and its elimination
kinetics were determined in tumor, liver, lung and plasma over a 6-day time period (n = 3/time
point) utilizing HPLC/MS-MS (Fig. 2A, B and Supplementary Table S4). Ganetespib is rapidly
distributed from the bloodstream into tissues and has a short half-life of 3 hours in plasma. The
half-life in normal liver and lung is 5-6 hours (Fig. 2A). In contrast, the half-life of ganetespib in
tumor was 58.3 hours, 10–19-fold longer than that in normal tissues or plasma, respectively (Fig.
2B). Additionally, at 144 hr after dosing, the tumor concentration of ganetespib remained 215-
fold higher than the median IC50 of 6.5 nmol/L required for antiproliferative cytotoxicity against
a broad NSCLC cell line panel (Supplementary Table S1).
The favorable intratumoral pharmacokinetics of ganetespib support evaluation of once-weekly
dosing. We therefore compared the relative efficacy of ganetespib and 17-AAG administered on
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a once per week schedule for three weeks against NCI-H1975 xenografts, utilizing a dose of 125
mg/kg ganetespib, and the HNSTD of 17-AAG of 175 mg/kg (Fig. 2C). Ganetespib displayed
significantly greater efficacy than 17-AAG, with the relative size of treated and control tumors of
15% and 50%, respectively (P < 0.05; one-way ANOVA), with no significant weight loss (Fig.
2D). Similar results were obtained with the HCC827 xenograft model when ganetespib was
administered once-weekly at the HNSTD (Supplementary Fig. S3).
Heterogeneous response of individual client proteins to HSP90 inhibition in vivo.
To assess the pharmacodynamic effects of ganetespib compared to 17-AAG in NCI-H1975
xenografts, we documented the kinetics of client depletion over a 6-day period following a single
intravenous administration at the HNSTD. The depletion of client kinases and the induction of
HSP70 and HSP27 were monitored by Western blotting of xenograft lysates (Fig. 3A) and
quantified by densitometry (Fig. 3B; n = 4/time point). Pharmacodynamic effects were
uniformly more pronounced in response to ganetespib than 17-AAG. In this model, where
EGFR depletion is critical, ganetespib depleted mutant EGFR (L858R/T790M) twice as
effectively than 17-AAG; for both drugs, peak suppression occurred at 24 hours post post-dose
(Fig. 3B). Surprisingly, recovery of EGFR expression was observed at later time points (Fig. 3B,
72 and 144 hours), despite the high intratumoral concentration of ganetespib (Fig. 2A). c-MET
and CDK4 depletion followed similar kinetics, although the recovery of c-MET expression was
slower than that of EGFR. In contrast, the depletion of other clients, such as c-RAF and AKT,
was gradual, without evidence of restoration of expression. Because ERBB2 is known to be
highly sensitive HSP90 client, the modest degree of depletion achieved in this experiment
prompted us to examine earlier time points, demonstrating that ERBB2 was rapidly depleted by
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6 hours, with a return to higher levels by 24 hours, although without restoration of baseline levels
as shown by the longer time course. ERBB2 was the most extensively depleted client at the
early time point (data not shown). The induction of the HSP70 and HSP27 chaperones in
response to ganetespib was as expected, reaching high levels by 72 hours; HSP70 induction
persisted until 144 hours, albeit with slight decline.
Immunohistochemical analyses of H1975 xenografts were also utilized to evaluate
pharmacodynamic changes after a single dose of ganetespib (Fig. 4). Confirming the Western
blot results (Fig. 3), a significant decrease in EGFR staining (P < 0.0025) was observed at 24
hours, but not at 6 hours, post-treatment (Fig. 4A). Additional multi-color staining, automated
image analysis and quantification demonstrated reduced proliferation and induction of apoptosis
at 24-48 hours post-dose, with recovery evident at 72 hours (Fig. 4B). In this mutant EGFR-
driven model, the kinetics of reduced BrdUrd incorporation and increased TUNEL staining
mirror those of EGFR depletion and recovery.
More frequent dosing improves the efficacy of ganetespib against the NCI-H1975 xenograft
model
Despite the favorable intratumoral pharmacokinetics of ganetespib supporting once-weekly
dosing, the depletion of mutant EGFR was not maintained through a 6-day period, suggesting
that more frequent dosing may be superior. To determine if this was the case, we compared the
schedules of 150 mg/kg administered once-weekly to 25 mg/kg administered five times weekly,
both over a three-week period (Fig. 5). More frequent administration of ganetespib resulted in
greater efficacy, with tumor regression achieved, rather than simply tumor growth inhibition. At
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day 29, compared to vehicle control, the relative tumor volume was 15% with once-weekly
dosing (consistent with the prior experiment in Fig. 2B), and -28% with five times weekly dosing
(P < 0.05; one-way ANOVA; Fig. 5A). Among the xenograft-bearing animals treated on the 5-
day schedule, all but one demonstrated tumor regression (Supplementary Fig. S4). Assessment
of body weight indicated that the once-weekly and 5-day schedules were equally well tolerated
(Fig. 5B).
Additionally, the pharmacodynamic effects of single dose and consecutive day dosing of
ganetespib were directly compared (Fig. 5C). Mice bearing NCI-H1975 xenografts were
administered a single dose of vehicle or ganetespib at 150 mg/kg, or alternatively vehicle or
ganetespib at 25 mg/kg x 5 consecutive days (with n = 3/timepoint for all vehicle or ganetespib
treated animals). After a single dose of ganetespib, mutant EGFR is depleted at 24 hours, with
expression restored by 72 hours. Downstream signaling, assessed with phospho-S6
immunohistochemistry, is also reduced at 24 hours, but reversing by 72 hours and fully restored
at 144 hours. Reductions in Ki-67 staining were seen at 24 and 72 hours, but were not
statistically significant. In contrast, when xenograft-bearing mice treated with ganetespib for 5
consecutive days were compared with those treated with vehicle, reductions in expression of
mutant EGFR, phospho-S6 and Ki-67 were seen throughout the 120-hour time course, extending
to 168 hours. Although several doses of ganetespib at 25 mg/kg are required to cause the degree
of reduction of mutant EGFR and phospho-S6 achieved 24 hours after a dose at 150 mg/kg, the
sustained pharmacodynamic effects with consecutive day dosing translate to superior anti-tumor
activity. Increases in HSP70 and HSP27 expression were observed after ganetespib exposure on
both schedules, consistent with HSP90 inhibition (Supplementary Figure S5).
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Ganetespib induces tumor regression in an ERBB2 YVMA–driven murine lung
adenocarcinoma model.
ERBB2 is one of the few HSP90 client proteins that demonstrated rapid depletion without full
re-expression after administration of a single dose of ganetespib (Fig. 3B). In isogenic Ba/F3
cells ectopically expressing ERBB2 harboring the YVMA exon 20 insertion activating mutation,
the most common ERBB2 kinase domain mutation identified, ganetespib demonstrated superior
activity compared with 17-AAG (Supplementary Fig. S6). These observations prompted us to
test the efficacy of ganetespib in a transgenic murine lung adenocarcinoma model driven by
ERBB2 YVMA (35). The no adverse effect level (NOAEL) dose was empirically determined at
25mg/kg three times per week in this model (data not shown). Compared to mice treated with
vehicle (n = 2), in ganetespib-treated mice (n = 4), there was statistically significant tumor
growth inhibition at 2 weeks, and reduction in tumor volume at 4 weeks (Fig. 6A), as
demonstrated by MRI scans (Fig. 6B). Immunohistochemical staining performed directly after
two doses 25 mg/kg ganetespib (administered every other day) demonstrated increased
expression of HSP27, consistent with HSP90 inhibition, and reduced expression of ERBB2. At
this early time point, phospho-S6 expression was also mildly decreased (Fig. 6C).
DISCUSSION
There is currently substantial interest in the development of HSP90 inhibitors for advanced
NSCLC, since many oncogenic drivers defining groups of adenocarcinomas are dependent on
HSP90 for conformational stability, including mutant EGFR, mutant ERBB2, EML4-ALK
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mutant BRAF, c-RAF and CDK4 (40), the latter two clients possibly underlying the sensitivity
of NSCLC cells carrying activating KRAS mutation (41, 42), demonstrated here with ganetespib
and previously with 17-AAG (43). We have shown that ganetespib binds to the N-terminus of
HSP90 and disrupts HSP90-p23 complexes, therefore resulting in inhibition of chaperone
activity and client protein depletion, which occurs with greater potency than with 17-AAG both
in vitro and in vivo. Among a large panel of genomically-defined NSCLC cell lines, including
those harboring EGFR mutation, ERBB2 mutation, ERBB2 amplification and KRAS mutation,
ganetespib routinely inhibited cellular proliferation with lower IC50 than 17-AAG. Additionally,
in ERBB-dependent xenograft and genetically engineered mouse models, ganetespib was well
tolerated, with activity at the NOAEL. Early phase clinical trials of ganetespib have
demonstrated that hepatic toxicity is substantially less common than with 17-AAG and its water
soluble derivatives (24, 44, 45); therefore, ganetespib may have improved therapeutic index
compared to agents in the geldanamycin class.
For NSCLC, the most mature HSP90 inhibitor studies have examined IPI-504 (retaspimycin
hydrochloride) (21) and ganetespib (46) in genomically-defined subsets of patients; in the latter
trial, ganetespib was used once weekly at the recommended phase 2 dose in cohorts of patients
whose tumors harbored mutant EGFR, mutant KRAS or wild type forms of both proteins. As
with IPI-504, the activity of ganetespib in the mutant EGFR arm was disappointing, with some
patients achieving either minor regression or disease stability lasting 12-16 weeks, but without
objective responses by response evaluation criteria in solid tumors (RECIST). The majority of
patients treated had acquired erlotinib resistance; although tumors harboring secondary T790M
mutation (9) or c-MET amplification (11) may be expected to respond, the activity of HSP90
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21
inhibition against tumors acquiring resistance by other mechanisms, including the emergence of
small cell histology or evidence of epithelial-mesenchymal transition has not been clarified (47).
In addition to the possible biological explanations for lack of response, our data suggest that the
schedule of drug administration may be critical. The preclinical pharmacokinetic profile of
ganetespib is typical of HSP90 inhibitors, demonstrating high penetrance and retention in tumor,
with short half-life in normal organs (18, 48). Nonetheless, the expression level of mutant
EGFR (L858R/T790M) in the NCI-H1975 xenograft model exhibits complete recovery by 5
days after single-dose exposure. These results suggest that once-weekly administration of
ganetespib will not be adequate to effectively suppress mutant EGFR/T790M signaling,
evidenced by the return of tumor cell proliferation and reversal of apoptosis that paralleled the
re-expression of mutant EGFR. Therefore, the sustained reduction in client protein expression
may be essential for efficient cell death in oncoprotein-driven NSCLC. Consistent with these
data, ganetespib was more efficacious in the NCI-H1975 xenograft model with daily x5 dosing,
which caused regressions rather than simply tumor growth inhibition. With consecutive day
dosing, there was prolonged depletion of the mutant EGFR client, with consequent extinguishing
of downstream signaling and proliferation. Importantly, an ongoing phase 1 trial of ganetespib
administered more than once per week will soon establish recommended phase 2 doses of both
twice-weekly and consecutive-day dosing schedules (45), with a plan to re-evaluate NSCLC
patients with tumors harboring EGFR mutation with these more frequent administration
schedules.
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Another strategy could be the combination of HSP90 inhibition and with a small molecule
inhibitor capable of suppression of the kinase activity of the reexpressed receptor. To date,
irreversible EGFR inhibitors have had only modest activity against EGFRs carrying T790M, but
may be adequate when combined with an HSP90 inhibitor.
The kinetics of c-MET and CDK4 depletion in response to ganetespib and 17-AAG in NCI-
H1975 xenografts were similar to those of EGFR, with a return of expression despite persistent
drug concentration in tumor, a phenomenon that has been observed with other HSP90 inhibitors
as well (48). These results suggest that there is a poor correlation between intratumoral drug
levels and the degree of HSP90 inhibition. The re-expression of these clients could therefore be
related to diminution in HSP90 inhibitory activity over time, secondary to altered intracellular
compartmentalization of drug, synthesis of new HSP90, or increased assembly of available
HSP90 into an active high-affinity, co-chaperone bound complex. Induction of the HSP70 and
HSP27 chaperones may also contribute to client re-expression.
However, not all clients are uniformly affected by such cellular changes. For example, in NCI-
H1975 cells, c-RAF continues to demonstrate gradual depletion after 17-AAG or ganetespib
exposure with lack of recovery of expression. Therefore, some clients may ultimately remain
sensitive to degradation, even if cellular HSP90 activity recovers to some extent in the presence
of drug.
Additionally, depending on cellular background, some clients exhibit exquisite sensitivity to
decreases in HSP90 activity with more rapid and complete depletion than others. This is the case
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23
with ERBB2 in NCI-H1975 cells, which was depleted by 6 hours; in addition, despite some
restoration of expression by 24 hours, levels of protein remained below baseline for a 6-day
period. A similar rapid decline of ERBB2 expression has been demonstrated with other HSP90
inhibitor compounds in ERBB2-amplified breast cancer cell lines and xenografts (48). Such may
also be true of EML4-ALK, which has been shown to be readily depleted from ALK-translocated
NSCLC cells by geldanamycins (13), to a greater degree than ERBB2 or EGFR are depleted
from ERBB2-amplified or EGFR-mutated breast and lung cancer cells, respectively (14). After a
single dose of IPI-504 administered to mice bearing ALK-translocated NCI-H3122 xenografts,
EML4-ALK levels were depleted in tumor for at least 48 hours; although longer time periods
were not examined, it is tempting to speculate that ALK would not be a client easily restored to
full levels of expression. These results may in part explain some of the successes of HSP90
inhibitor compounds to date, including 17-AAG in ERBB2-amplified breast cancer (19) and IPI-
504 and weekly ganetespib in ALK-rearranged NSCLC, where both drugs have produced durable
partial responses (21, 46).
In addition to ganetespib, several other non-geldanamycin compounds are under active
development. Currently, ganetespib is distinguished from several of these compounds since it
lacks ocular toxicities, with more favorable retinal distribution and elimination (44, 45, 49-51).
AT13387, discovered with a fragment-based discovery approach, has also been characterized in
NCI-H1975 NSCLC cells (52). In vitro, a 7-hour exposure resulted in depletion of mutant
EGFR lasting in excess of 168 hours. Following single dose exposure to mice bearing NCI-
H1975 xenografts, there was rapid clearance from blood with prolonged intratumoral retention of
drug to 240 hours; however, similar to ganetespib, depleted mutant EGFR expression with
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24
downstream signaling was restored by 72 hours (52). An administration schedule on days 1, 4,
8, 12 and 16 led to similar tumor growth inhibition as a once-weekly schedule, neither producing
clear regressions, raising the possibility that consecutive day dosing schedules may be optimal in
this model as well as in trials in which NSCLC patients with tumors harboring EGFR mutation
are evaluated. To this end, once-weekly, twice-weekly and consecutive day dosing
administration schedules of AT13387 are all under evaluation in Phase 1 trials (49, 53).
NVP-AUY922, an isoxazole resorcinol, has been studied in multiple preclinical models. A wide
variety of NSCLC cell lines, including those harboring EGFR mutation, are highly sensitive to
NVP-AUY922, with low nanomolar IC50’s in 72-hour MTS assays (54). In vivo, AUY922 is
also preferentially retained in tumor over plasma (55). In ERBB2-dependent BT-474 breast
cancer xenografts, ERBB2 depletion occurred by 6 hours after a single dose, with restoration of
expression by 48 hours (56). More sustained regression was noted with three times per week
compared to once-weekly administration, at the expense of significantly greater toxicity,
manifesting with animal weight loss. Because substantial tumor growth inhibition was still noted
with once-weekly dosing, NVP-AUY922 has been evaluated clinically with this schedule (56).
Interestingly, in a Phase 2 NSCLC trial, confirmed partial responses were noted in patients with
ALK-positive tumors, but also among 5 of 35 patients with tumors harboring EGFR mutation
(57). The kinetics of EGFR depletion in response to NVP-AUY922 in preclinical EGFR-
dependent NSCLC models will therefore be of substantial interest in order to explain the
preliminary efficacy of once-weekly dosing in this subset.
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The efficient and prolonged depletion of ERBB2 in xenografts following HSP90 inhibitor
exposure, and the substantial superiority of ganetespib over 17-AAG against Ba/F3 cells
transformed to IL-3 independence by ERBB2 carrying an exon 20 YVMA activating insertion
mutation, prompted us to evaluate ganetespib in a mouse model of lung adenocarcinoma driven
by the same mutation. Previously, we showed that these tumors demonstrate only partial
sensitivity to a dual EGFR-ERBB2 tyrosine kinase inhibitor that is augmented by mTOR
inhibition, which further extinguishes the ERBB2-driven signaling pathway (35). In this model,
single agent ganetespib demonstrated anti-tumor activity with depletion of mutant ERBB2 after
initial exposure, translating to tumor growth inhibition predominating after 2 weeks of treatment,
followed by tumor regressions after 4 weeks of drug exposure. These results suggest potential
efficacy of ganetespib against NSCLCs driven by mutant ERBB2, a group not yet represented
among ganetespib-treated patients whose tumors express wild type EGFR and KRAS.
In summary, ganetespib displays improved preclinical potency compared to 17-AAG with
potential for activity in several NSCLC subsets defined by their addiction to individual
oncoproteins. In particular, the current results justify further clinical evaluation of ganetespib in
ERBB family member-driven tumors. Optimization of dosing schedules and integration with
tyrosine kinase inhibitor based therapy, both of which may vary depending on the targeted client,
remain important research avenues that will move the HSP90 inhibitor field forward.
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26
GRANT SUPPORT
This work was supported by the Dana-Farber/Harvard Cancer Center Specialized Program of
Research Excellence (SPORE) in Lung Cancer, NIH Grant P50 CA90578 (K.K.W and G.I.S.), a
Career Development Award as part of the SPORE (T.S.), and a Sponsored Research Agreement
from Synta Pharmaceuticals (K.K.W. and G.I.S.).
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27
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FIGURE LEGENDS
Figure 1. Ganetespib binds to HSP90, disrupts HSP90-p23 complexes, depletes client
proteins and suppresses signaling cascades with greater potency than 17-AAG in NSCLC
cell lines. (A) NCI-H1975 lysates were treated with DMSO (0 μmol/L) or the indicated
concentrations of 17-AAG or ganetespib and then incubated with biotinylated geldanamycin
(biotin-GM) linked to Streptavidin-containing beads. The amount of HSP90 bound to biotin—
GM in the absence or presence of drug was determined by Western blot. (B) NCI-H1975 lysates
from cells treated with DMSO or the indicated concentrations of 17-AAG or ganetespib for 2
hours were subjected to immunoprecipitation with anti-p23 antibody followed by Western
blotting with the indicated antibodies. (C) Exponentially growing NCI-H1975 (EGFR
L858R/T790M), cells were treated with DMSO (0 μmol/L) or the indicated concentrations of 17-
AAG or ganetespib for 24 h, and lysates were subjected to Western blotting with the indicated
antibodies, demonstrating depletion of receptor tyrosine kinases and suppression of downstream
signaling in response to HSP90 inhibition. (D) HCC827 cells were treated with DMSO (0
μmol/L) or the indicated concentrations of ganetespib for 24 h. Lysates were subject to Western
blotting with indicated antibodies, demonstrating induction of pro-apoptotic Bim.
Figure 2. Ganetespib accumulates in tumor relative to normal tissues and displays greater
in vivo efficacy than 17-AAG. (A) Mice bearing NCI-H1975 xenografts received a single
intravenous dose of 125 mg/kg ganetespib and concentrations were determined in tumor, liver,
lung and plasma over a 24 hr time period. (B) Similar analysis over of ganetespib
concentrations after a single intravenous dose over a 144 hr time period. In (A) and (B), N =
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36
3/time point; error bars represent +/- SEM. (C) Mice bearing NCI-H1975 xenografts were
treated intravenously 1X/week for 3 weeks (arrowheads) with vehicle, 17-AAG (175 mg/kg) or
ganetespib (125 mg/kg) Tumor volumes were assessed over the one-month time course. Error
bars +/- SEM. (D) For mice treated in (C), cumulative average changes in body weight were
followed over the one-month time course. Error bars +/- SEM.
Figure 3. Depletion of HSP90 clients in vivo in response to 17-AAG and ganetespib.
(A) Mice bearing NCI-H1975 xenografts model, received a single dose of vehicle, 17-AAG
(175 mg/kg) or ganetespib (125 mg/kg). At 24, 72 and 144 hrs, lysates from harvested
xenografts (N = 4/group) were subjected to Western blotting with the indicated antibodies. (B)
Western blots were quantified using ImageJ and expression levels are presented as a percentage
of expression in vehicle-treated tumors. Error bars +/- SEM.
Figure 4. Ganetespib inhibits proliferation and induces apoptosis in parallel with EGFR
depletion in NCI-H1975 xenografts. (A) Mice bearing NCI-H1975 xenografts were treated
with a single intravenous dose of vehicle or 125 mg/kg ganetespib. Six and 24 hours post-
treatment, cryosections of harvested xenografts (N = 8/group) were subjected to
immunohistochemistry for EGFR. (Left) Representative images. (Right) Quantification of
EGFR staining at 6 and 24 hrs post-treatment. Error bars + SEM. (B) Mice bearing NCI-H1975
xenografts were treated as in A; cyrosections from tumors (N = 8/group) harvested at 6, 24, 48
and 72 hours after treatment were subjected to immunohistochemistry for markers of
proliferation (BrdU), apoptosis (TUNEL), vascular density (CD31), hypoxia (pimonidazole) and
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37
perfusion (DiOC7(3)). (Left) Representative images. (Right) Quantification of BrdU and
TUNEL staining at the indicated time points post-treatment. Error bars + SEM.
Figure 5. Comparison of once-weekly and five times per week dosing of ganetespib.
(A) Mice bearing NCI-H1975 xenografts were treated with vehicle, 150 mg/kg ganetespib once
weekly (white arrows) or 25 mg/kg ganetespib five times per week (black arrows) over a three-
week period. % T/C values are shown. * P < 0.05. Errors bars + SEM. (B) For mice treated in
(A), the percent change in body weight was followed over the treatment time course. Errors bars
+ SEM. (C) Mice bearing NCI-H1975 xenografts were treated with vehicle or ganetespib at 150
mg/kg x 1, with xenografts harvested 24, 72 and 144 hours after dosing (V1-24, V1-72, V1-144;
G1-24, G1-72 and G1-144, for vehicle and ganetespib-treated samples, respectively).
Alternatively, mice were treated with vehicle or ganetespib at 25 mg/kg/day x 5 days, with
xenografts harvested 24, 72, 120 and 168 hours after the first dose (i.e. the 120- and 168-hour
time points were 24 and 72 hours after the last dose; V5-24, V5-72, V5-120, V5-168; G5-24, G5-
72, G5-120 and G5-168 for vehicle and ganestespib-treated samples, respectively). For each
time point and treatment, xenografts were subjected to immunohistochemistry with the indicated
antibodies and quantified as described in the Materials and Methods. (Left) Graphs represent the
percent change in average IHC positivity of ganetespib-treated xenografts (n = 3) relative to the
corresponding vehicle controls (n =3). Bars, Standard Error. * P < 0.05 for the comparison of
ganetespib-treated xenografts compared to the corresponding vehicle control. (Right)
Representative immunohistochemically stained sections, demonstrating more sustained
reductions in expression of mutant EGFR, phospho-S6 and Ki-67 with consecutive day dosing.
Magnification, 100X. Bar, 100 μm.
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Figure 6. Ganetespib induces tumor regression in a mouse lung carcinoma model driven
by ERRB2YVMA. (A) ERBB2 YVMA mice were treated with doxycycline for 4 weeks and
imaged before treatment (baseline) and after every other day treatment with vehicle (V) or 25
mg/kg ganetespib (G) at 25 mg/kg for two or four weeks, after which tumor volume was
calculated, demonstrating tumor growth inhibition at 2 weeks and regression at 4 weeks in
ganetespib treated mice. (Left) Quantification of tumor volumes in individual mice. (Right)
Results averaged over the entire vehicle or gantespib-treated group. * P < 0.05 compared to
vehicle-treated contol. (B) Representative MRI scans performed at baseline and at 2 and 4
weeks post-treatment, demonstrating substantial reduction in tumor burden in two of the
ganetespib–treated mice. (C) HER2 YVMA tumor-bearing mice were left untreated, or treated
with vehicle or ganetespib (25 mg/kg every other day for two doses) and sacrificed for histology
and immunohistochemical analyses. Representative results for the peripheral compartment are
shown. Magnification 400x. Scale bars 50 μm. (D) Immunohistochemical quantification for
showing percentage of positive cells. Error bars + SEM. * P < 0.001.
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Published OnlineFirst July 17, 2012.Clin Cancer Res Takeshi Shimamura, Samanthi A. Perera, Kevin P. Foley, et al. Non-Small Cell Lung Cancerhas Potent Antitumor Activity in In Vitro and In Vivo Models of Ganetespib (STA-9090), a Non-Geldanamycin HSP90 Inhibitor,
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