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Translational Cancer Mechanisms and Therapy

Consecutive Day HSP90 Inhibitor AdministrationImproves Efficacy in Murine Models of KIT-DrivenMalignancies and Canine Mast Cell TumorsCheryl A. London1,2, Jaime Acquaviva3, Donald L. Smith3, Manuel Sequeira3,Luisa Shin Ogawa3, Heather L. Gardner4, Louis Feo Bernabe1, Misty D. Bear1,Sandra A. Bechtel5, and David A. Proia3

Abstract

Purpose: STA-1474, prodrug of the heat shock protein 90inhibitor (HSP90i) ganetespib, previously demonstratedactivity in canine preclinical models of cancer; interestingly,prolonged infusions were associated with improved biologicactivity. The purpose of this study was to identify the idealtreatment schedule for HSP90i in preclinical models of KIT-driven malignancies and in dogs with spontaneous mast celltumors (MCT), where KIT is a known driver.

Experimental Design: In vitro andmurine xenograft experi-ments and clinical studies in dogs with MCTs were used todefine the effects of HSP90i-dosing regimen on client proteindownregulation and antitumor activity.

Results: Continuous HSP90 inhibition led to durabledestabilization of client proteins in vitro; however, transientexposure required >10� drug for comparable effects. In vivo,KIT was rapidly degraded following a single dose of HSP90i

but returned to baseline levels within a day. HSP90 levelsincreased and stabilized 16 hours after HSP90i and were notelevated following a subsequent near-term exposure, pro-viding a functional pool of chaperone to stabilize proteinsand a means for greater therapeutic activity upon HSP90ireexposure. HSP90i administered on days 1 and 2 (D1/D2)demonstrated increased biologic activity compared with D1treatment in KIT or EGFR-driven murine tumor models. Ina trial of dogs with MCT, D1/D2 dosing of HSP90i wasassociated with sustained KIT downregulation, 50% objec-tive response rate and 100% clinical benefit rate comparedwith D1 and D1/D4 schedules.

Conclusions: These data provide further evidence thatprolonged HSP90i exposure improves biologic activitythrough sustained downregulation of client proteins. ClinCancer Res; 24(24); 6396–407. �2018 AACR.

IntroductionHeat shock proteins (HSP) are molecular chaperones integral

for regulating multiple aspects of posttranslational proteinhomeostasis for numerous client proteins, including proteinkinases and transcription factors involved in cell proliferation,signaling, and oncogenesis (1, 2). Heat shock protein 90 (HSP90)is important for maintenance and stabilization of known onco-proteins, including KIT, MET, and EGFR, among others. Upregu-lation of HSP90 expression and function has been well describedin cancer cells (3–5), and its activity is generally considered crucialfor tumor cell survival, supporting its targeting for therapeuticintervention.

Considerable progress in the development of HSP90 inhi-bitors (HSP90i) has occurred over the past 15 years. Earlygeldanamycin-based HSP90i displayed modest activity andwere associated with significant clinical toxicity, as well aspotential multidrug efflux and formulation problems, limitingtheir clinical development (6–9). STA-1474 (Synta Pharmaceu-ticals Corp) is a novel water-soluble resorcinol-containingcompound metabolized to STA-9090, a potent HSP90i thatbinds to the N-terminus of the HSP90 ATP-binding domain,resulting in destabilization and degradation of HSP90 clientproteins. HSP90i have shown activity against several types ofcancers both in vitro and in vivo, including models of caninemast cell tumor (MCT) and canine osteosarcoma (10, 11).STA-9090 (ganetespib) demonstrated activity in a canine MCT

1Departments of Veterinary Biosciences and Clinical Sciences, The Ohio StateUniversity, Columbus, Ohio. 2Cummings School of Veterinary Medicine, TuftsUniversity, Grafton, Massachusetts. 3Synta Pharmaceuticals, Lexington, Massa-chusetts. 4Sackler School of Graduate Biomedical Sciences, Tufts University,Boston, Massachusetts. 5Department of Veterinary Medicine and Surgery,College of Veterinary Medicine, University of Missouri, Columbia, Missouri.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for J. Acquaviva: Astellas Institute for Regenerative Medicine,Marlborough, Massachusetts; current address for D.L. Smith: Charles RiverLaboratories, Worcester, Massachusetts; current address for M. Sequeira:Charles River Laboratories, Worcester, Massachusetts; current address forL.S. Ogawa: Proteostasis Therapeutics, Cambridge, Massachusetts; currentaddress for L.F. Bernabe: Hospital Ars Veterinaria, Barcelona, Spain; currentaddress for M.D. Bear: Kansas State University, College of Veterinary Medicine,Manhattan, Kansas; current address for S.A. Bechtel: Department of SmallAnimal Clinical Sciences, College of Veterinary Medicine, University of Florida,Gainesville, Florida; and current address for D.A. Proia: C4 Therapeutics,Watertown, Massachusetts.

C.A. London and J. Acquaviva contributed equally to this article.

Prior presentation: Presented in abstract atAACR 104thAnnualMeeting; April 6–10, 2013; Washington, DC.

CorrespondingAuthor:CherylA. London, TuftsUniversity, 200WestboroRoad,Cummings School, North Grafton, MA 01536. Phone: 614-915-7409; Fax: 508-839-7922; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0703

�2018 American Association for Cancer Research.

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mouse xenograft model and induced growth inhibition, apo-ptosis, and downregulation of pKIT, KIT, and AKT in both KIT-dependent and KIT-independent tumors (10).

More recently, in a phase I clinical trial, STA-1474 wasadministered to 25 dogs with spontaneous tumors exploring3 different dosing schemes (12). Measurable objectiveresponses were observed in dogs with MCT (n ¼ 3), osteosar-coma, melanoma, and thyroid carcinoma (n ¼ 1 each), for anobjective response rate of 24% (6/25). Data from this trialsuggested that longer drug infusion times (8 hours vs. 1 hour)were potentially associated with greater biologic activity, evi-denced by a higher objective response rate and sustainedplasma levels of STA-9090. When administered over 8 hours,STA-9090 plasma levels were maintained for approximately 12hours, compared with only 5 to 6 hours when administered as a1-hour infusion. Low-objective response rates have beenreported in phase I and II human clinical trials of STA-9090in which drug was administered as a 1-hour infusion once ortwice per week (72 hours apart), although several patientsexperienced stable disease (13–15). Taken together, these datasuggest that longer plasma exposures of STA-9090 may beassociated with greater biologic activity. It is well establishedthat inhibition of HSP90 induces the heat shock response,whereby HSF-1 is released from HSP90 and induces the syn-thesis of HSP90 and other heat shock proteins. Thus, onepotential mechanism for the improved efficacy with longerinfusion times may be a result of inhibition of this newlysynthesized material.

Given the clinical challenges associated with prolongedintravenous infusions, this study sought to determine whether2 consecutive days of treatment with STA-1474 was superior toother dose regimens with respect to target modulation andclinical response to therapy by providing a strategy to inhibitboth baseline HSP90 as well as HSP90i-induced HSP90. Assuch, the overriding objective of this study was to expand uponthe initial phase I study findings and identify the optimaldosing schedule of STA-1474 in dogs with spontaneous MCT,using both response to therapy and duration of KIT down-regulation as biomarkers of efficacy.

Materials and MethodsCell lines, antibodies, and reagents

The H1975 and Kasumi cell lines were obtained from theAmerican Type Culture Collection and maintained according tostandard techniques and used within 2 years of receipt. Primaryantibodies were obtained from Cell Signaling Technology, withthe exception of GAPDH (Santa Cruz Biotechnology) andHSP70(Enzo life sciences). Ganetespib and STA-12-7888 (D3-ganete-spib) were synthesized by Synta Pharmaceuticals Corp.

Western blottingFollowing in vitro assays, tumor cells were disrupted in lysis

buffer (Cell Signaling Technology) on ice for 10 minutes. For thepharmacodynamic analysis, xenograft tumors (average volume of100–200mm3)were excised, cut in half, and flash frozen in liquidnitrogen. Each tumor fragment was lysed in 0.5 mL of lysis bufferusing a FastPrep-24 homogenizer and Lysing Matrix A (MPBiomedicals). Lysates were clarified by centrifugation and equalamounts of protein resolved by SDS-PAGE before transfer tonitrocellulose membranes (Invitrogen). Membranes wereblocked with Starting Block T20 Blocking Buffer (Thermo FisherScientific) and immunoblotted with the indicated antibodies.Antibody–antigen complexes were visualized using an Odysseysystem (LI-COR).

HSP90-binding assayH1975 cells, cultured in RPMI-1640 and 10% FBS, were

seeded at a density of 3 � 105 cells per well in 6-well plates.Twenty-four hours later, cells were treated with ganetespib asindicated and incubated at 37�C. Cells were washed twice incold PBS then lysed in cold HSP90-binding buffer (20 mmol/LHEPES pH 7.3, 1 mmol/L EDTA, 100 mmol/L KCl, 5 mmol/LMgCl, 0.01% v/v NP-40, 0.5 mg/mL bovine gamma globulin,1 mmol/L TCEP) by incubation on ice for 10 minutes followedby three freeze/thaw cycles. Lysates were clarified by centrifu-gation at 14,000 � g. To remove unbound ganetespib, lysateswere passed over 40 K MWCO size exclusion columns (ThermoFisher Scientific). To titrate unoccupied HSP90-binding sites,10 mmol/L of a deuterated form of ganetespib (D3-ganetespib)was added to eluates and incubated at 4�C for 2 hours thenpassed over a size exclusion column to remove unboundD3-ganetespib. Total protein from flow through was quantifiedby BCA protein assay and all samples diluted to 1 mg/mL. Theconcentrations of ganetespib and D3-ganetespib were mea-sured by LC/MS-MS. A Phenomenex Kinetex column (C18,30 � 2.1 mm, 2.6 mm) was used with a run time of 3.5 minutesper sample. The following equation was used to calculate thepercentage of ganetespib bound to HSP90 (HSP90 occupancy):(ganetespib)/[(ganetespib) þ (D3-ganetespib)] � 100.

In vivo xenograft tumor modelsFemale CB.17 (SCID) mice (Charles River Laboratories) at

7 to 12 weeks of age were maintained in a pathogen-freeenvironment and all in vivo procedures were approved by theSynta Pharmaceuticals Corp. Institutional Animal Care andUse Committee. Human GIST882 cells were provided byDr. Jonathan Fletcher (Dana-Farber Cancer Institute) andimplanted subcutaneously at 10 � 106 into mice. Mice bearingestablished tumors (�110 mm3) were randomized into treat-ment groups of 8 and dosed intravenously (i.v.) with vehicle or

Translational Relevance

Heat shockprotein 90 (HSP90) is crucial formaintenance ofseveral known oncoproteins. The resorcinol-containingHSP90 inhibitor STA-9090 (ganetespib) has demonstratedsuperior antitumor activity in vitro and in preclinical in vivomodels compared with geldanamycin compounds; however,certain client proteins, such as KIT, are only transiently down-regulated after HSP90 blockade. A prior study in dogs foundthat sustained blood levels of ganetespib from 200 to600 ng/mL maintained over 8 hours were associated withenhanced biologic activity. This study investigated four dose-equivalent treatment regimens in dogs with spontaneousmastcell tumors using response to therapy and KIT downregulationas biomarkers of efficacy and found that target modulationwas maximized using a 2-day consecutive dosing regimen.These findings support the notion that efficacy of HSP90inhibition is dependent on maximizing target modulationthrough optimized drug regimens.

Improved Efficacy with Consecutive Day HSP90i Dosing

www.aacrjournals.org Clin Cancer Res; 24(24) December 15, 2018 6397




ganetespib, formulated in DRD (10% DMSO, 18% CremophorRH 40, 3.6% dextrose), using the schedules indicated. HumanH1975 non–small cell lung cancer (NSCLC) cells were pur-chased from the ATCC, selected to stably express a HIF1a-LUCreporter and implanted at 10 � 106 into mice. Mice bearingestablished tumors (�143 mm3) were randomized into treat-ment groups of 4 and dosed intravenously with vehicle organetespib, formulated in DRD, using the schedules indicated.Tumor volumes (V) were calculated by caliper measurements ofthe width (W), length (L) and thickness (T) of each tumor usingthe formula: V ¼ 0.5236 (LWT). Tumor growth inhibition wasdetermined as described previously (16). Statistical analyseswere conducted using two-way ANOVA followed by Bonferroniposttests.

Clinical trial designA phase II randomized open label study of STA-1474 was

completed in client-owned dogs with spontaneous MCT greaterthan 2 cm in size. Dogs were randomized to receive a single doseof STA-1474 intravenously at 6 mg/kg over 1 hour (Cohort A), 6mg/kg over 8 hours (cohort B), 3 mg/kg over 1 hour on day 1 andday 2 (D1/D2) (cohort C), or 3mg/kg onday 1 andday 4 (D1/D4;cohort D). This treatment schedule was repeated for a total of 4cycles. Tumor biopsies, obtained under sedation and local anes-thesia using a 3 to 5mmpunchbiopsy or 14-gauge Tru-cut needle,were taken at baseline prior to the first treatment with STA-1474and then 24 and 72 hours after the last treatment of the first cycle.All biopsy samples were flash frozen in liquid nitrogen and storedat �80�C until analysis. Routine hematologic and biochemicalprofiles were performed once weekly throughout the study peri-od. Dogs were evaluated for clinical toxicities and adverse eventsat each study visit. Clinical toxicities related to disease progressionor other unrelated comorbid conditions were not consideredadverse events (AE). All AEs were graded in accordance withestablished VCOG-CTCAE criteria (17).

Eligibility and ethics statementTheClinical Research andAdvisingCommittee at theCollege of

Veterinary Medicine and the Institutional Animal Care and UseCommittee at The Ohio State University approved this study.Informed consent was obtained from all owners prior to studyentry. Tobe considered for enrollment, dogswere required tohavea confirmed diagnosis of MCT. Additional required eligibilitycriteria included the following: at least 1 year of age at the time ofenrollment; adequate organ function as indicated by standardlaboratory evaluation; no prior chemotherapy administration orradiotherapy; and no other serious systemic disorder incompat-ible with the study. Prior use of prednisone was permitted.

STA-1474 FormulationSTA-1474was supplied by Synta Pharmaceuticals in 30mg/mL

vials containing either 30 mg or 150 mg of drug. Unopened vialswere stored for no longer than 30 days at 4�C and protected fromlight. STA-1474 was diluted in Plasmalyte-148 in a 250 mLinfusion bag for administration and was used within 24 hoursof preparation. Each vial was used within 96 hours of sealpuncture, and the remaining drug was discarded.

Tumor response assessmentResponse assessments were performed at enrollment and once

weekly thereafter. Responses were characterized according to the

VCOG response evaluation criteria for solid tumors in dogs (v1.0)via serial examination (18). An objective response consisted ofeither a complete or partial response. A complete response (CR)was defined as complete resolution of all target lesions. Partialresponse (PR) was defined as �30% reduction in the sum ofthe longest dimensions of the target lesions, taking as a referencethe baseline sum of the longest dimensions. Progressive disease(PD) was defined as a >20% increase in the sum of the longestdimensions of the target lesions, taking as a reference the small-est sum of the longest dimensions since treatment initiation, orthe appearance of at least 1 new nontarget lesion. Stable disease(SD) was defined as neither sufficient decrease nor increase intarget lesions to be considered an objective response or diseaseprogression, respectively. All target lesions, including metastaticlesions, were evaluated using caliper measurements or ultrasono-graphic evaluation of distant metastasis.

Concomitant medicationsThe primary AEs associated with STA-1474 administration

identified in the phase I trial were gastrointestinal in nature(12). Therefore, dogs were administered maropitant, ondanse-tron, and metronidazole prophylactically before and after eachSTA-1474 infusion. Concomitant medications used to preventand/or treat clinical toxicities were used at the discretionof the attending clinician and included the following: anti-emetics (maropitant, ondansetron, metoclopramide, mirtaza-pine), gastroprotectants (omeprazole, famotidine, sucralfate,pantoprazole), intravenous fluids, antidiarrheals (metronidazole,loperamide, bismuth subsalicylate, probiotics), antihistamines(diphenhydramine), analgesics (tramadol, butorphanol),hepatoprotectants (S-adenosylmethionine, silybin), antibiotics(amoxicillin clavulanate, cephalexin, doxycycline, cefovecin).Prednisone use was permitted during the study if dogs werealready receiving the drug and had demonstrated PD despitetreatment, and as a single administration for relief of swellingand erythema associated with MCT biopsy.

Immunoprecipitation and Western blotting of canine biopsiesMCT biopsies were collected and flash frozen before treat-

ment (0 hour), and at 24 and 72 hours after completion of theSTA-1474 i.v. infusion. Frozen biopsies were pulverized into apowder while in liquid nitrogen. Tissue powder was thawedbriefly on ice, then resuspended in fresh lysis buffer consistingof 20 mmol/L Tris-HCl pH 8.0, 137 mmol/L NaCl, 10%glycerol, 1% IPEGAL CA-630, 10 mmol/L ethylenediaminete-traacetic acid (EDTA), 1 mg/mL aprotinin, 1 mg/mL leupeptin,1 mg/mL pepstatin A, 1 mmol/L phenylmethylsulphonyl fluo-ride, 1 mmol/L sodium orthovanadate, and 10 mmol/L sodiumfluoride (all from Sigma-Aldrich). Samples were vortexed, thenrocked for 1 hour at 4�C. The protein lysates were collectedand quantified using the Bradford assay. Protein (1 mg/mL persample) was precleared for 1 hour with protein A agarose beads(Roche Diagnostic Corp.), followed by immunoprecipitationwith 5 mg of anti-KIT (CD117, Pierce/Thermo Fisher Scientific).Immunoprecipitated protein was separated by SDS-PAGE andtransferred to a PVDF membrane. Membranes were incubatedovernight with anti-KIT antibody (CD117, Pierce/ThermoFisher Scientific). The membranes were incubated for 1 hourwith goat anti-rabbit horseradish peroxidase linked secondaryantibody, washed, and exposed to substrate (SuperSignal WestDura Extended Duration Substrate, Pierce).

London et al.

Clin Cancer Res; 24(24) December 15, 2018 Clinical Cancer Research6398




ResultsConstant ganetespib exposure inhibits HSP90 client proteinsin oncogene-driven cell lines

In vitromodeling of ganetespib exposure was used to provideinsight into the mechanism of action associated with improvedbiological responses after prolonged HSP90i exposure in vivo.EGFR-mutant (L858R and T790M) H1975 NSCLC cells wereexposed to increasing doses of ganetespib for either 1 hour or24 hours (continuous), followed by a washout and incubationwith media and DMSO vehicle for 23 hours. Nearly completeclient protein turnover (EGFR, HER2, MET), effector inactiva-tion (pAKT, pSTAT3, pERK), heat shock response (HSP70),and signals of apoptosis (BIM) were observed with constantganetespib treatment at 50 nmol/L (Fig. 1A). Approximately30-fold higher ganetespib (1,500 nm) concentration wasrequired to obtain comparable effects after a 1-hour treatment.Similar results were observed in the acute myeloid leukemia

line Kasumi-1, which harbors an activating mutation (N822K)in KIT (Fig. 1B), and in H2228 NSCLC cells, H3122 NSCLCcells, SKBR3 breast cancer cells, A375 melanoma cells, andMKN45 gastric cancer cells (data not shown).

We next assessed the kinetics of protein degradation follow-ing robust HSP90 inhibition and observed variability amongproteins, consistent with normal variability in rates of proteinturnover. EGFR (L858R T790M) and MET showed �50%turnover after 8 hours of exposure, and nearly 100% turnoverby 24 hours in H1975 NSCLC cells (Fig. 1C), concordant withtheir published half-lives (19–21). Rapid HER2 turnoverwas observed, with the majority of turnover observedwithin 4 hours. Consistent with its published half-life, HER2turnover was nearly complete by 8 hours (22). KIT expressionin Kasumi-1 cells appeared to follow this trend, with signifi-cant degradation of total and phosphorylated KIT at 4 hours(Fig. 1D), reflecting the published half-lives for the two

Figure 1.

Effects of ganetespib on HSP90 client protein turnover in vitro. H1975 cells (A) and Kasumi cells (B) were exposed to increasing concentrations ofganetespib, either continuously or for 1 hour, followed by drug washout. Lysates were prepared 24 hours later and immunoblotted with the indicatedantibodies. H1975 (C) and Kasumi (D) cells were treated with vehicle or ganetespib (100 nmol/L) for the indicated times. Lysates were immunoblottedwith the indicated antibodies.






isoforms (23). Taken together, these results indicate that dura-ble inhibitory effects on the chaperone require either contin-uous low levels of drug or pulse-dosed high drug levels.

Duration of HSP90 client protein degradation varies afterganetespib administration

Therapeutic activity of HSP90 inhibition is likely directlyrelated to the time taken for client proteins to return topretreatment expression levels after their degradation. As such,the pharmacodynamics of receptor tyrosine kinase expressionin both H1975 and Kasumi-1 cell line xenografts were inves-tigated. EGFR, HER2, and MET suppression was observed inH1795 xenografts 24 hours after ganetespib exposure andpersisted for approximately 5 days, (Fig. 2A). HSP70 levelsincreased following ganetespib exposure and remained elevat-ed after 5 days. Phosphorylated KIT (Y719) was rapidly degrad-ed (6 hours) in gastrointestinal stromal (GIST) xenografttumors expressing an activating KIT mutation (K642E) as wasthe cyclin-dependent kinase CDC2/CDK1 (Fig. 2B). In contrastto EGFR, HER2, MET, and CDC2/CDK1, expression of KIT andlevels of pKIT (Y719) returned to near baseline levels within24 hours after ganetespib administration. These data suggestthat in diseases driven by proteins such as EGFR and HER2,weekly treatment with an HSP90i may yield clinical activitywhereas in tumors driven by proteins such as KIT, efficacy couldbe minimal and more frequent dosing or longer exposure timeswould likely be required for optimal biologic activity.

Daily consecutive HSP90 inhibitor administration does notpromote a secondary induction of HSP90 synthesis

Why did KIT levels stabilize so quickly after HSP90 inhibition?We hypothesized that the observed rapid stabilization of KITafter HSP90i treatment likely had to be HSP90-dependent. More-over, given the feedback upregulation of HSP70 followingganetespib exposure (Figs. 1 and 2) perhaps HSP90 upregulationwas compensating for the loss of steady state HSP90 activity.We assessed the kinetics of HSP90 expression following gane-tespib exposure in vitro. Shown in Fig. 2C, HSP90 was induced inH1975 cells after treatment with a single dose of ganetespib,reaching a maximum at 8 hours and remaining elevated at 24hours. Based on these results, we expected the percentage of totalHSP90 bound by ganetespib following a single pulse dose todecline over time because it would be diluted by the newlysynthesized, unbound HSP90. To evaluate this, we first deter-mined the amount of ganetespib required to saturate availableHSP90-binding sites, by treating H1975 cells for 1 hour withincreasing doses of ganetespib, followed by the addition of adeuterated form of ganetespib (10 mmol/L) to the cell lysate tooccupy any unbound sites, an approach similar to that previouslyreported (24). Approximately 64 to 128 nmol/L ganetespib wassufficient to occupy �80% of available binding sites (Fig. 2D)within 5 to 10 minutes of drug exposure (Fig. 2E). These valuesare approximate as we did not correct for binding to the plate,cells, or proteins in/out of the cell. Next, we investigated whetherthe total number of HSP90-binding sites would increase withtime following ganetespib exposure, based on total HSP90ioccupancy. At 6 hours after ganetespib treatment, there was anegligible rise in occupancy, but by 16 hours, the number ofbinding sites increased 2.3-fold (Fig. 2F). This occupancy couldbe blocked with the protein synthesis inhibitor cycloheximide.Taken together, these results suggest that although an initial

exposure to ganetespib can effectively saturate HSP90-bindingsites, upregulation of new HSP90 protein provides a new pool ofchaperone; combined with drug clearance (25), this results in asustained increase in HSP90 expression.

We then asked whether 2 ganetespib treatments, adminis-tered 24 hours apart, would trigger another round of HSP90synthesis. Although a slight rise in HSP70 expression wasobserved between the 24-hour single exposure and 8 hoursafter 2 consecutive-day treatments (Fig. 2C), levels of HSP90remained stable and were not greatly elevated by the secondganetespib treatment, nor was there a substantial rise in newHPS90-binding sites (Fig. 2H). These data support the notionthat the capacity to make new HSP90 following HSP90i treat-ment may be limited with a subsequent, near-term exposure toHSP90i and may provide a strategy for greater HSP90 occu-pancy, more durable client protein degradation, and ultimate-ly, enhanced efficacy.

To test whether a second, near-term administration of ganete-spib would limit the capacity to make new HSP90, ganetespiblevels were measured in H1975 cell lysates (after size exclusioncolumn to remove free drug) 4 hours after a single 1-hour treat-ment or after 2 consecutive days of treatment. Indeed, ganetespiblevels were 1.6-fold higher in the latter treatment group (Fig. 2G).HSP90 occupancy by ganetespib in H1975 cells (Fig. 2H) fol-lowing a single pulse exposure decreased 44% between 8 and24 hours in relation to the total level of HSP90i (ganetespibplus deuterated ganetespib), corresponding with an increase innew HSP90 synthesis (Fig. 2C). Consistent with new HSP90synthesis, ganetespib occupied an even greater total number ofHSP90-binding sites after 2 consecutive days of dosing; however,these levels remained stable for 48 hours. These results indicatethat consecutive ganetespib administration does not promote asecondary induction of HSP90 synthesis and represents a viablemechanism to increase total HSP90 binding/inhibition.

Daily consecutive ganetespib administration is associated withbiologic activity in murine xenografts

Mice bearing human GIST882 tumor xenografts weretreated with vehicle or ganetespib administered as a singledose (100 mg/kg), q12h (50 mg/kg), or on 2 consecutive days(50 mg/kg). These doses have been well documented as safe inmice (below the highest nonseverely toxic dose) and exposuresare consistent with what is clinically obtainable and meaningfulin humans (14, 25). Tumors initially regressed in all drug treat-ment groups; however, progression was evident early in the onceweekly dosing arm. More frequent ganetespib exposure resultedin biologic activity (SD, PR) at the end of the 2-week study;however, the difference was not statistically significant (Fig.3A). Similar results were observed in H1975 xenografts (Fig.3B), where 2 cycles of once weekly ganetespib treatment resultedin SD and 2 cycles of 2 consecutive-day ganetespib treatmentpromoted significant and durable tumor regression (PR; P ¼0.001). These findings are consistent with previously publisheddata demonstrating SD with weekly ganetespib treatment inH1975 tumors and regression when dosed 5 times per week (25).

Validation of 2-day dosing regimen in spontaneous caninemodel of KIT dysregulation

We had previously evaluated the water-soluble prodrugof ganetespib, STA-1474, in dogs with spontaneous cancersand found that biologic activity was greatest in dogs that

London et al.





Figure 2.

Two consecutive-day dosing as a strategy to saturate baseline and ganetespib-induced HSP90 binding sites. A and B, Pharmacodynamic assessment ofHSP90 client turnover. A, Mice bearing H1975 xenografts were dosed once with vehicle or ganetespib (150 mg/kg), tumors were harvested at theindicated times postdose, and lysates were immunoblotted with the indicated antibodies. B, Mice bearing Kasumi xenografts were dosed once withvehicle or ganetespib (150 mg/kg), tumors were harvested at the indicated times postdose, and lysates were immunoblotted with the indicated antibodies.C, Comparison of HSP90 induction for day 1 (D1) versus D1/D2 dosing regimens in vitro. For D1 dosing, H1975 cells were treated with ganetespib (100 nmol/L)for 1 hour followed by drug washout. For D1/D2 dosing, a second dose of ganetespib was added to cells 24 hours after D1 drug washout. Lysates wereprepared at the indicated times postdose and immunoblotted with the indicated antibodies. For quantification, HSP70 and HSP90 levels werenormalized to GAPDH loading control. D and E, HSP90-binding sites are rapidly occupied by ganetespib in a dose-dependent manner in H1975 cells. D, Cellswere treated with increasing concentrations of ganetespib for 24 hours. E, Binding kinetics were assessed by treating cells with ganetespib (100 nmol/L) forthe indicated times. The amount of ganetespib bound to HSP90 was quantified using the competitive HSP90-binding assay. Results plotted aspercentage of total HSP90-binding sites occupied by ganetespib. F, Kinetics of ganetespib-induced HSP90-binding sites and dependence on proteinsynthesis. Cells were treated with ganetespib (100 nmol/L) � cycloheximide (1 mg/mL) for the indicated times, followed by HSP90-binding assay. Thefold change in HSP90-binding sites was calculated from the total amount of drug (ganetespib þ D3-ganetespib) detected after saturation of available HSP90-binding sites. G, Cellular levels of ganetespib in D1 versus D1/D2 dosing. Cells were treated with ganetespib (100 nmol/L) using D1 and D1/D2 dosingschedules, and the total amount of ganetespib was measured 4 hours postdose following size exclusion filtration. H, Assessment of degree anddurability of HSP90-binding site occupancy with single and two consecutive-day dosing. Cells were treated with ganetespib (100 nmol/L) using D1 and D1/D2dosing schedules. HSP90-binding assays were performed at the indicated time postdose. Results plotted as percentage of total HSP90-binding sitesoccupied by ganetespib.






received an 8-hour drug infusion. This correlated to sustained(8þ hours) plasma ganetespib levels ranging from 200 to600 ng/mL, and we hypothesized that this was sufficient topromote additional inhibition of any newly synthesized HSP90following initial drug inhibition. However, a similar protocolcould not be used in humans, given the solvent requiredfor ganetespib, thus limiting infusion times in humans. There-fore, we initiated a clinical trial in dogs with spontaneousMCT to determine whether D1/D2 treatment with an HSP90i(STA-1474) was superior to other dose regimens with respectto objective responses and target modulation.

Clinical trial regimen and demographics. Twenty-four dogs (n ¼ 6per treatment group) with MCT were enrolled into this clinicaltrial. All MCTs were chemotherapy and radiotherapy na€�ve.Patient demographics are detailed in Table 1. The median agewas 8 years, and the median weight was 34.1 kg. Eleven dogsreceived a short course of corticosteroids (typically one injec-tion or 2–3 days of therapy) during the study period due toswelling associated with MCT biopsies. In mice, short-termcorticosteroid use does not affect KIT expression in normalmast cells (26). In dogs with MCT treated with prednisonealone at 40 mg/m2 daily, total KIT expression levels did notchange based on IHC performed at baseline and day 21 oftherapy (27). These data suggest that the short-term use ofcorticosteroids in this study was unlikely to have affected theassessment of changes in KIT expression following treatment.

Treatment groups. Treatment groups were designed to expandupon the phase I study results to determine whether 2 consecutive

days of STA-1474 treatment were superior to one 8-hour infusion,with respect to target modulation and objective response totherapy. Two cohorts of dogs (cohorts A and B) were treated at6 mg/kg STA-1474 over 1 hour or 8 hours (n ¼ 6 each) once perweek. An additional 2 cohorts of dogs (n¼ 6 each; cohorts C andD) were treated at 3 mg/kg STA-1474 over 1 hour on days 1 and 2(D1/D2), or days 1 and 4 (D1/D4). Each dog was scheduled toreceive 4 cycles of STA-1474. Three dogs were withdrawn from thestudy prior to completion (n¼ 1 comorbid condition; n¼ 3 PD).

Adverse events. AEs associated with STA-1474 infusion weremild and primarily gastrointestinal in nature, consisting ofdiarrhea, vomiting, anorexia, and lethargy (Table 2; Supple-mentary Table S1). There were no dose-limiting toxicities, defin-ed as any grade 3 or 4 adverse event, related to STA-1474administration. Liver transaminase elevations occurred in alldose groups. These elevations were considered related to steroidadministration or comorbid conditions and were not consid-ered related to STA-1474 administration. Low-grade BUN eleva-tions and anemia occurred in cohorts A, B, and D. These clinicaltoxicities are well established in dogs with MCT disease andwere deemed unlikely to be related to STA-1474. One dog withpreexisting IRIS stage II chronic kidney disease (CKD) devel-oped grade 3 elevations in BUN and creatinine during thestudy period. This dog was hospitalized on fluid therapy for3 days and was discharged from the hospital upon resolution ofthe azotemia. This dog was subsequently withdrawn from theclinical trial due to progressive CKD.

Other treatment emergent AEs included regurgitation andhind limb weakness in one dog (cohort C). This dog

Figure 3.

Dosing b.i.d. or 2 consecutive days results in greater therapeutic activity compared with once weekly treatment. A, SCID mice bearing establishedhuman GIST882 tumors (n ¼ 8 mice/group) were administered 2 weekly cycles of vehicle or ganetespib, i.v., as indicated starting on day 22. Data areexpressed as mean tumor volumes � SEM. Treatment/control values (T/C), as a measure of percent tumor growth inhibition or regression, were asfollows: 48% for ganetespib dosed 1�/week; �2.2% for ganetespib dosed D1/D2; 0.4% for ganetespib dosed b.i.d. All doses were well tolerated withthe exception of a single animal in the ganetespib b.i.d. group whose body weight dropped to �21% on day 31 and rapidly recovered. B, SCID micebearing established human H1975 tumors (n ¼ 4 mice/group) were administered 2 weekly cycles of vehicle or ganetespib, i.v., as indicated, starting onday 14. Data are expressed as mean tumor volumes � SEM. T/C values were as follows: �13% for ganetespib dosed 1�/week; �53% for ganetespibdosed D1/D2; �61% for ganetespib dosed b.i.d. All doses were well tolerated during the course of the study. �� , P ¼ 0.001; ns, not significant forstatistics between once weekly and D1/D2 ganetespib groups.

London et al.





regurgitated a small amount of water 1 day after the firstinfusion, and metoclopramide was administered prophylacti-cally for subsequent STA-1474 infusions. At week 3, the samedog was reportedly weak in the hind legs when ambulating athome. No detectable neurologic dysfunction or weakness wasdetectable while in the hospital or during STA-1474 treatments.The weakness was determined to be most likely related toobesity; however, an adverse event secondary to STA-1474could not be excluded.

Response to therapy. Measurable clinical benefit was observ-ed in all cohorts; however, no durable (�5 week) objectiveresponses were seen in dogs that received STA-1474 at 6 mg/kgonce weekly over 1 hour (Fig. 4A). Three dogs were withdrawnprior to study completion due to PD (n ¼ 2 cohort D; n ¼ 1cohort A), and 1 dog (cohort B) was removed from thestudy due to progression of CKD. Dogs that received 3 mg/kgSTA-1474 on 2 consecutive days demonstrated biologicactivity (100%) with 50% of those dogs experiencing an

objective response, when compared with all other dosinggroups (Fig. 4A and C).

Evaluation of canine tumor biopsies. Immunoprecipitation andWestern blotting were performed on flash frozen tumor samplesfrom 3 dogs each in cohort taken predose and 24 and 72 hoursafter STA-1474 dosing (Fig. 4B). Although some degree of KITmodulation was observed in all cohorts, only those samples fromcohort C demonstrated durable loss of KIT that persisted at the72-hour time point. These data are consistent with the clinicalresponses and confirming the notion that durable HSP90 inhi-bition following treatment with STA-1474 requires more sus-tained drug exposures.

DiscussionA variety of HSP90 inhibitors have demonstrated activity

and promise in vitro; however, translating this into clinical benefitin human cancer patients has proven difficult, highlighting

Table 1. Patient demographics

A B C DCohort 6 mg/kg, 1 hour 6 mg/kg, 8 hours 3 mg/kg, 1 hour D1/D2 3 mg/kg, 1 hour D1/D4 All dogs

Number of dogs 6 6 6 6 24Age (years)Median 8 8.5 10.5 6 8Range 2–12 6–14 7–12 5–10 2–14

Weight (kg)Median 36.95 21.15 34.15 33.95 34.1Range 24.3–50.4 3.3–39.2 9.6–47.8 29.4–37.7 3.3–50.4

GenderMale intact 1 0 0 1 2Male castrated 4 3 3 1 11Female intact 0 0 0 0 0Female spayed 1 3 3 4 11

BreedMixed breed 4 3 2 1 10Labrador retriever 1 1 1 2 5Boxer 1 0 1 2 4Golden retriever 0 0 1 1 2Other 0 2 1 0 3

Tumor gradeGrade 2 5 5 3 3 16Grade 3 1 3 2 6

Unknown 1 1 2MetastasisLymph node 1 2 1 3 7Distant metastasis 0 0 0 1 1

PrednisoneYes 3 2 4 2 11No 3 4 2 4 13

Table 2. Treatment emergent gastrointestinal AEs

Grade of AEsVomiting Diarrhea Anorexia Weight loss Lethargy

Dose (mg/kg) and regimen No. dogs 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

6 mg/kg 6 1 3 61 hour6 mg/kg 6 1 1 4 1 5 38 hours3 mg/kg 6 1 1 1 4 11 hour, D1/D23 mg/kg 6 1 1 1 1 1 4 11 hour, D1/D4






the challenges associated with inhibition of this pathway in atherapeutically efficacious manner. Several mechanisms havebeen proposed for the observed limited biologic activity inpatients, including concurrent upregulation of cochaperones thatstabilize client proteins, low tumor reliance on the client protein,and suboptimal client protein inhibition due to conventionaldosing regimens (6). This study used a relevant large animalmodel of a KIT-driven tumor, recapitulating the molecular het-erogeneity and environmental context of HSP90 inhibition inhuman cancers, to validate an improved dosing schedule, iden-

tified through preclinical work in human and murine cell lineswith known KIT dysregulation, associated with objectiveresponses and modulation of a driver oncoprotein (KIT) follow-ing STA-1474 treatment. The use of preclinical models withdistinct tumor histology but with a similar molecular aberrationstrengthens the preclinical evidence used to determine the opti-mal dosing regimen of HSP90i treatment.

In cancer cell lines, the dose and regimen of HSP90i treatmenthas a significant impact on the degree and duration of clientprotein degradation. Consistent with drug concentrations

Figure 4.

Administration schedule affectsclinical activity and target modulationin canine MCT. A, Waterfall plotshowing percent change in tumor sizefor each subject at the end of the studyperiod. Partial responses (�30%reduction) are illustrated by the blackdotted line. Diagonal marks denotepatients with PD at week 4 due toprogression of nontarget lesions. B,Tumor biopsy samples at 0, 24, and 72hours after STA-1474 treatment wereanalyzed by immunoprecipitation andWestern blotting using anti-KITantibody as indicated. The blotscorresponding to individual patientresponses in the waterfall plot aboveare indicated. C, Response to therapyfollowing consecutive-day treatment(cohort C).

London et al.





necessary to saturate chaperone binding sites, constant exposureto ganetespib (50–125 nmol/L) promoted degradation of HSP90clients and inactivation of downstream effectors. In contrast,transient ganetespib treatment required 1–2 log more drug forcomparable effects. This may be due in part to the need to occupyand inhibit nascent HSP90 induced by the heat shock response,requiring nonspecifically retained excess drug to act as a drugreservoir.

Variations in client turnover and stabilization were observedin murine xenograft models following ganetespib treatment.For example, rapid turnover of pKIT was observed followingsingle-dose ganetespib administration in mice bearing GISTxenograft tumors. However, expression levels of the kinasereturned to near baseline levels 24 hours after ganetespibadministration, resulting in poor tumor control in vivo inanimals receiving once weekly dosing. Similar pKIT kineticswere observed in patient tumor biopsies during a phase II trialof ganetespib administered at the maximum tolerated dosed(200 mg/m2) once weekly for 3 out of 4 weeks (28). Althoughdisease stabilization was observed in 12/23 patients, durableinhibition of pKIT and its downstream pathways was notobserved, supporting our preclinical finding that transientsuppression of KIT activation occurs with once weekly HSP90iadministration.

In contrast, mutant EGFR was fully degraded 24 hours afterganetespib administration in the H1975 NSCLC tumors andremained at low levels for the duration of the 5-day study. Inthis model, weekly ganetespib treatment led to SD. However, CRswere observed in transgenic tumor models, dosing daily with aselective third-generation EGFR inhibitor, suggesting that con-stant inhibition is required for objective responses (29). Indeed,administering low-dose ganetespib 5 days per week resulted insignificant tumor regression in H1975 xenograft models (25).Interestingly, lung tumor xenografts driven by an EML4-ALKfusion were highly responsive to once weekly ganetespib treat-ment, with objective responses observed at theMTD (150mg/kg)and SD observed at lower doses (50 mg/kg) (30).

We sought to identify an HSP90i administration schedulethat would combine a clinically feasible dosing regimen withthe biologic activity of constant HSP90i exposure to inhibitboth baseline and drug-induced HSP90, thus extending theduration of client protein degradation. It is firmly establishedthat inhibition of HSP90 triggers a heat shock response, result-ing in the induction of heat shock proteins, including HSP90and HSP70. Our occupancy studies demonstrated that ganete-spib could rapidly occupy the majority of cellular HSP90 inshort-term studies, supporting previously published researchregarding the impact HSP90i have on oncoprotein stability andcell signaling pathways. Importantly, the rise in new HSP90-binding sites after HSP90i exposure was determined to be dueto new protein synthesis, and subsequent HSP90i administra-tion occupied both baseline and induced HSP90-binding sites.These effects result in increased ganetespib exposure, andtherefore efficacy, as evidenced by the responses observed inthe NSCLC and GIST xenograft studies.

The importance of HSP90 inhibitor dose schedule is highlight-ed in recent clinical studies. For example, a phase II trial of theHSP90 inhibitor 17-AAG (tanespimycin) was associated withminimal HSP90 inhibition and few objective responses in met-astatic melanoma (9). Pharmacodynamic results from this studyindicated that the limited success was attributable to suboptimal

dose schedule, evidenced by limited inhibition of target clientproteins and variable pharmacokinetics (9). More recently, aphase I study of STA-1474 in spontaneous canine cancers sug-gested that prolonged infusions were associated with greaterbiologic activity (12). In the present study, two 1-hour infusionsgiven 24 hours apart was associated with the greatest objectiveresponse rate (50%), supporting the notion that sustainedHSP90inhibition is associated with improved biologic activity. Thistreatment regimen was also associated with a persistent decreasein KIT in tumor samples 72 hours posttreatment, while lessfrequent STA-1474 administrationwas associatedwith fewer dogsexperiencing clinical benefit and persistent KIT expression intumor samples after treatment. These findings warrant futureprospective studies in larger cohorts of patients to validate theresults presented herein.

Taken together, our data underscore the importance of deter-mining the dosing regimens that best modulate intratumoralHSP90 function to provide sustained inhibition of cancer cellgrowth and survival. AEs reported herein were consistent withthose reported in the phase I study of STA-1474 in dogs withcancer (12). AEs were primarily gastrointestinal in nature(lethargy, anorexia, vomiting, diarrhea) and resolved withsupportive care. EGFR is an HSP90 client protein with a rolein intestinal epithelial integrity, and EGFR inhibition has beenpostulated to be a mechanism of diarrhea associated withHSP90 inhibitors (31–33). Gastrointestinal AEs are the knownprimary toxicity of STA-1474, and in the present study, pro-phylactic management of nausea and diarrhea were utilized tohelp reduce the incidence and severity of gastrointestinal toxi-cities. Importantly, all dosing regimens were well tolerated inthis study, and enhanced toxicity was not apparent due toincreased frequency of STA-1474 administration. This is par-ticularly important with respect to geldanamycin derivatives, inwhich the administration schedule significantly affects drugtolerability (34–36).

In addition, ganetespib has demonstrated synergistic activitywith the MET kinase inhibitor crizotinib, both in the setting ofcrizotinib-sensitive and -resistant MET-driven tumor models(37). These findings raise the question of whether a combi-natorial approach to treatment of KIT-driven malignanciescould produce synergistic activity and abrogate the develop-ment of drug resistance. Although the objective responsesobserved in this study after STA-1474 treatment demonstratethe potential therapeutic utility of HSP90 inhibition in canineMCT, no dogs experienced a CR to therapy, and furtherinvestigation of combinatorial approaches may improveobjective responses.

In summary, more frequent exposure to STA-1474 showsevidence of improved biologic activity without an enhancedtoxicity profile in a relevant, spontaneous, large animal modelof cancer. These findings indicate that more frequent exposureto HSP90 inhibitors will translate into improved biologic activityin people.

Disclosure of Potential Conflicts of InterestD.A. Proia was Director at Synta Pharmaceuticals. No potential conflicts of

interest were disclosed by the other authors.

Authors' ContributionsConception and design: C.A. London, D.A. ProiaDevelopment of methodology: D.A. Proia






Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.A. London, J. Acquaviva, D.L. Smith, M. Sequeira,L.S. Ogawa, L.F. Bernabe, M.D. Bear, S.A. BechtelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.A. London, J. Acquaviva, D.L. Smith, D.A. ProiaWriting, review, and/or revision of themanuscript:C.A. London, J. Acquaviva,H.L. Gardner, S.A. Bechtel, D.A. ProiaAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): C.A. London, H.L. Gardner, L.F. Bernabe,M.D. Bear, S.A. BechtelStudy supervision: C.A. London, D.A. Proia

AcknowledgmentsThe Veterinary Clinical Research Services Shared Resource at the Ohio

State University College of Veterinary Medicine coordinated all aspects ofthis study including generation of case report forms, collection of samples,collation of data, quality assurance, coordination of other clinical sites, andfinal quality control on all data from all study sites. This project wassupported by the following grants: UL1TR001070 from the National Center

for Advancing Translational Sciences and P30CA016058 from the NationalCancer Institute to The Ohio State University. Synta Pharmaceuticals pro-vided funding for this clinical trial. The content is solely the responsibility ofthe authors and does not necessarily represent the official views of theNational Center for Advancing Translational Sciences, National CancerInstitute or the National Institutes of Health, Synta Pharmaceuticals, orAldeyra Therapeutics. This clinical trial was directly supported by funds fromSynta Pharmaceuticals. This project was supported by the following grants:UL1TR001070 from the National Center for Advancing TranslationalSciences and P30CA016058 from the National Cancer Institute to The OhioState University.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 1, 2018; revised July 3, 2018; accepted August 27, 2018;published first August 31, 2018.

References1. Trepel J, Mollapour M, Giaccone G, Neckers L. Targeting the dynamic

HSP90 complex in cancer. Nat Rev Cancer 2010;10:537–49.2. Nathan DF, Vos MH, Lindquist S. In vivo functions of the Saccharo-

myces cerevisiae Hsp90 chaperone. Proc Natl Acad Sci U S A 1997;94:12949–56.

3. Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat RevCancer 2005;5:761–72.

4. Kamal A, Thao L, Sensintaffar J, Zhang L, BoehmMF, Fritz LC, et al. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90inhibitors. Nature 2003;425:407–10.

5. Duus J, Bahar HI, Venkataraman G, Ozpuyan F, Izban KF, Al-Masri H, et al.Analysis of expression of heat shock protein-90 (HSP90) and the effects ofHSP90 inhibitor (17-AAG) in multiple myeloma. Leuk Lymphoma2006;47:1369–78.

6. Neckers L, Workman P. Hsp90 molecular chaperone inhibitors: are wethere yet? Clin Cancer Res 2012;18:64–76.

7. Banerji U, O'Donnell A, Scurr M, Pacey S, Stapleton S, Asad Y, et al. Phase Ipharmacokinetic and pharmacodynamic study of 17-allylamino,17-demethoxygeldanamycin in patients with advanced malignancies.J Clin Oncol 2005;23:4152–61.

8. Pacey S, Gore M, Chao D, Banerji U, Larkin J, Sarker S, et al. A Phase IItrial of 17-allylamino, 17-demethoxygeldanamycin (17-AAG, tanespi-mycin) in patients with metastatic melanoma. Invest New Drugs2012;30:341–9.

9. SolitDB,Osman I, PolskyD, Panageas KS,DaudA,Goydos JS, et al. Phase IItrial of 17-allylamino-17-demethoxygeldanamycin in patients with met-astatic melanoma. Clin Cancer Res 2008;14:8302–7.

10. Lin TY, Bear M, Du Z, Foley KP, Ying W, Barsoum J, et al. The novel HSP90inhibitor STA-9090 exhibits activity against Kit-dependent and -indepen-dent malignant mast cell tumors. Exp Hematol 2008;36:1266–77.

11. McCleese JK, Bear MD, Fossey SL, Mihalek RM, Foley KP, YingW, et al. Thenovel HSP90 inhibitor STA-1474 exhibits biologic activity against osteo-sarcoma cell lines. Int J Cancer 2009;125:2792–801.

12. London CA, Bear MD, McCleese J, Foley KP, Paalangara R, Inoue T, et al.Phase I Evaluation of STA-1474, a Prodrug of the Novel HSP90Inhibitor Ganetespib, in Dogs with Spontaneous Cancer. PLoS One2011;6:e27018.

13. Jhaveri K, Chandarlapaty S, LakeD,Gilewski T, RobsonM,Goldfarb S, et al.A phase II open-label study of ganetespib, a novel heat shock protein90 inhibitor for patients with metastatic breast cancer. Clin Breast Cancer2014;14:154–60.

14. Goldman JW, Raju RN, Gordon GA, El-Hariry I, Teofilivici F, VukovicVM, et al. A first in human, safety, pharmacokinetics, and clinicalactivity phase I study of once weekly administration of the Hsp90inhibitor ganetespib (STA-9090) in patients with solid malignancies.BMC Cancer 2013;13:152.

15. Cercek A, Shia J, GollubM,Chou JF, CapanuM, Raasch P, et al. Ganetespib,a novel Hsp90 inhibitor in patients with KRAS mutated and wild type,

refractory metastatic colorectal cancer. Clin Colorectal Cancer 2014;13:207–12.

16. Proia DA, Foley KP, Korbut T, Sang J, Smith D, Bates RC, et al. Multifacetedintervention by the Hsp90 inhibitor ganetespib (STA-9090) in cancercells with activated JAK/STAT signaling. PLoS One 2011;6:e18552.

17. Veterinary cooperative oncology group - common terminology criteriafor adverse events (VCOG-CTCAE) following chemotherapy or biologicalantineoplastic therapy in dogs and cats v1.1. Vet Comp Oncol. 2016;14:417–46.

18. Nguyen SM, Thamm DH, Vail DM, London CA. Response evaluationcriteria for solid tumours in dogs (v1.0): a Veterinary CooperativeOncology Group (VCOG) consensus document. Vet Comp Oncol2015;13:176–83.

19. Giordano S, Di Renzo MF, Narsimhan RP, Cooper CS, Rosa C, ComoglioPM. Biosynthesis of the protein encoded by the c-met proto-oncogene.Oncogene 1989;4:1383–8.

20. Seth D, Shaw K, Jazayeri J, Leedman PJ. Complex post-transcriptionalregulation of EGF-receptor expression by EGF and TGF-alpha in humanprostate cancer cells. Br J Cancer 1999;80:657–69.

21. Greig MJ, Niessen S, Weinrich SL, Feng JL, Shi M, Johnson TO. Effects ofActivating Mutations on EGFR Cellular Protein Turnover and Amino AcidRecycling Determined Using SILAC Mass Spectrometry. Int J Cell Biol2015;2015:798936.

22. Ren XR, Wei J, Lei G, Wang J, Lu J, Xia W, et al. Polyclonal HER2-specificantibodies induced by vaccination mediate receptor internalization anddegradation in tumor cells. Breast Cancer Res 2012;14:R89.

23. Yee NS, Hsiau CW, Serve H, Vosseller K, Besmer P. Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidy-linositol 3'-kinase, and protein kinase C. J Biol Chem 1994;269:31991–8.

24. Tillotson B, Slocum K, Coco J, Whitebread N, Thomas B, West KA, et al.Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinantof client protein degradation and tumor growth arrest in vivo. J Biol Chem2010;285:39835–43.

25. Shimamura T, Perera SA, Foley KP, Sang J, Rodig SJ, Inoue T, et al.Ganetespib (STA-9090), a nongeldanamycin HSP90 inhibitor, has potentantitumor activity in in vitro and in vivo models of non-small cell lungcancer. Clin Cancer Res 2012;18:4973–85.

26. Bankova LG, Dwyer DF, Liu AY, Austen KF, GurishMF. Maturation of mastcell progenitors to mucosal mast cells during allergic pulmonary inflam-mation in mice. Mucosal Immunol 2015;8:596–606.

27. Teng SP, Hsu WL, Chiu CY, Wong ML, Chang SC. Overexpression ofP-glycoprotein, STAT3, phospho-STAT3 and KIT in spontaneouscanine cutaneous mast cell tumours before and after prednisolone treat-ment. Vet J 2012;193:551–6.

28. Demetri GD, Heinrich MC, Chmielowski B, Morgan JA, George S, BradleyR, et al. An open-label phase II study of the Hsp90 inhibitor ganetespib(STA-9090) in patients (pts) with metastatic and/or unresectable GIST.J Clin Oncol 2011;29:10011–10011.

London et al.





29. Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ,et al. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediatedresistance to EGFR inhibitors in lung cancer. Cancer Discov 2014;4:1046–61.

30. Sang J, Acquaviva J, Friedland JC, Smith DL, Sequeira M, Zhang C, et al.Targeted inhibition of the molecular chaperone Hsp90 overcomes ALKinhibitor resistance in non-small cell lung cancer. Cancer Discov2013;3:430–43.

31. Abud HE, Watson N, Heath JK. Growth of intestinal epithelium inorgan culture is dependent on EGF signalling. Exp Cell Res 2005;303:252–62.

32. Suzuki A, Sekiya S, Gunshima E, Fujii S, Taniguchi H. EGF signalingactivates proliferation and blocks apoptosis of mouse and human intes-tinal stem/progenitor cells in long-termmonolayer cell culture. Lab Invest2010;90:1425–36.

33. Shimamura T, Lowell AM, Engelman JA, Shapiro GI. Epidermalgrowth factor receptors harboring kinase domain mutations asso-ciate with the heat shock protein 90 chaperone and are destabi-

lized following exposure to geldanamycins. Cancer Res 2005;65:6401–8.

34. Solit DB, Ivy SP, Kopil C, Sikorski R, Morris MJ, Slovin SF, et al. Phase I trialof 17-allylamino-17-demethoxygeldanamycin in patients with advancedcancer. Clin Cancer Res 2007;13:1775–82.

35. Grem JL, Morrison G, Guo XD, Agnew E, Takimoto CH, Thomas R, et al.Phase I and pharmacologic study of 17-(allylamino)-17-demethoxygel-danamycin in adult patients with solid tumors. J Clin Oncol 2005;23:1885–93.

36. Ramanathan RK, Egorin MJ, Erlichman C, Remick SC, Ramalingam SS,Naret C, et al. Phase I pharmacokinetic and pharmacodynamic study of 17-dimethylaminoethylamino-17-demethoxygeldanamycin, an inhibitor ofheat-shock protein 90, in patientswith advanced solid tumors. J ClinOncol2010;28:1520–6.

37. Miyajima N, Tsutsumi S, Sourbier C, Beebe K, Mollapour M, Rivas C, et al.The HSP90 inhibitor ganetespib synergizes with the MET kinase inhibitorcrizotinib in both crizotinib-sensitive and -resistant MET-driven tumormodels. Cancer Res 2013;73:7022–33.






2018;24:6396-6407. Published OnlineFirst August 31, 2018.Clin Cancer Res Cheryl A. London, Jaime Acquaviva, Donald L. Smith, et al. Tumorsin Murine Models of KIT-Driven Malignancies and Canine Mast Cell Consecutive Day HSP90 Inhibitor Administration Improves Efficacy

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