Ganetespib (STA-9090), a Non-Geldanamycin HSP90 Inhibitor, has … · KIT-dependent mast cell tumors (26), MET-dependent osteosarcoma cell lines (27), Wilms tumor 1 (WT1)-dependent

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




8

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




9

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




11

(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




12

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 >




14

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




15

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




16

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




17

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




18

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




20

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




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.




22

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




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




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.




25

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.




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.).




27

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35

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 =




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




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.




38

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.






















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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