the hsp90 inhibitor, at13387, is effective against …...preclinical development the hsp90...

Preclinical Development

The HSP90 Inhibitor, AT13387, Is Effective againstImatinib-Sensitive and -Resistant GastrointestinalStromal Tumor Models

Tomoko Smyth1, Thomas Van Looy2, Jayne E. Curry1, Ana M. Rodriguez-Lopez1, Agnieszka Wozniak2,Meijun Zhu3, Rachel Donsky3, Jennifer G. Morgan3, Mark Mayeda3, Jonathan A. Fletcher3, Patrick Sch€offski2,John Lyons1, Neil T. Thompson1, and Nicola G. Wallis1

AbstractThe majority of gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT, an

HSP90 client protein. Further secondary resistance mutations within KIT limit clinical responses to tyrosine

kinase inhibitors, such as imatinib. The dependence of KIT and its mutated forms on HSP90 suggests that

HSP90 inhibitionmight be a valuable treatment option for GIST,whichwould be equally effective on imatinib-

sensitive and -resistant clones. We investigated the activity of AT13387, a potent HSP90 inhibitor currently

being evaluated in clinical trials, in both in vitro and in vivoGISTmodels. AT13387 inhibited the proliferation of

imatinib-sensitive (GIST882, GIST-T1) and -resistant (GIST430, GIST48) cell lines, including those resistant to

the geldanamycin analogueHSP90 inhibitor, 17-AAG. TreatmentwithAT13387 resulted in depletion ofHSP90

client proteins, KIT and AKT, along with their phospho-forms in imatinib-sensitive and -resistant cell lines,

irrespective of KIT mutation. KIT signaling was ablated, whereas HSP70, a marker of HSP90 inhibition, was

induced. In vivo, antitumor activity of AT13387 was showed in both the imatinib-sensitive, GIST-PSW,

xenograft model and a newly characterized imatinib-resistant, GIST430, xenograft model. Induction of HSP70,

depletionof phospho-KITand inhibitionofKIT signalingwere seen in tumors frombothmodels after treatment

with AT13387. A combination of imatinib and AT13387 treatment in the imatinib-resistant GIST430 model

significantlyenhanced tumorgrowth inhibitionover eitherof themonotherapies. Importantly, the combination

ofAT13387 and imatinibwaswell tolerated. These results suggestAT13387 is an excellent candidate for clinical

testing in GIST in combination with imatinib. Mol Cancer Ther; 11(8); 1799–808. �2012 AACR.

IntroductionGastrointestinal stromal tumors (GIST) are mesenchy-

mal tumors that are frequently characterized by expres-sion of the receptor tyrosine kinase KIT (1, 2). About 85%of advanced GISTs have activating mutations in KIT,whereas another 5% harbor mutations in platelet-derivedgrowth factor receptor alpha (PDGFRA; ref. 3). Typicallyfirst-line treatment for inoperable GIST is imatinib, but20%of patientswithGISTdonot respond to this treatmentwith response apparently related, in part, to the type and

position of the primary activating mutation (4, 5). Thosethat respond ultimately develop resistance to treatmentlargely through secondary mutations in KIT (6). Resis-tance tends to be highly heterogeneous with differentmutations observed within the same patient at differentmetastatic sites and even within the same tumor nodule(7). The second-line treatment, sunitinib, is active againstsomeof the imatinib-resistant formsofKIT, but eventuallythe vastmajority of patientswill also develop resistance tosunitinib. Because of the extensive inter- and intralesionalheterogeneity of resistance mutations within individualpatients it has been suggested that tyrosine kinase inhi-bitors (TKI) are unlikely to have curative potential in thisdisease (2).

KIT and PDGFRA are clients for HSP90, a key chaper-one and an attractive target in many cancers (2, 8, 9).Mutated forms of the proteins are also dependent on thechaperone for their stability (10). It has been suggestedthat use of HSP90 inhibitors could be a good strategy fortreatment in GIST, as they would target both imatinib-sensitive and -resistant GIST, irrespective of the type ofKIT mutation (11). Inhibition of HSP90 has previouslybeen shown to bring about degradation of KIT, withthe geldanamycin analogues 17-AAG, IPI-493, and

Authors' Affiliations: 1Astex Pharmaceuticals, Cambridge, United King-dom; 2Department of General Medical Oncology and Laboratory of Exper-imental Oncology, University Hospitals Gasthuisberg, Catholic Universityof Leuven, Leuven, Belgium; and 3Department of Pathology, Brigham andWomen's Hospital, Boston, Massachusetts

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Tomoko Smyth, Astex Pharmaceuticals, 436Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, UnitedKingdom. Phone: 44-0-1223-435017; Fax: 44-0-1223-226201; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-1046

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on June 26, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst June 19, 2012; DOI: 10.1158/1535-7163.MCT-11-1046

http://mct.aacrjournals.org/

retaspimycin (IPI-504) having shownantitumor activity ina number ofGIST andmast cell tumormodels (10–14). Thegeldanamycins have been extensively studied and testedin the clinic, however they have clinical limitations (15).17-AAGhas shownhepatotoxicity and formulation issueswhile a phase III GIST trial with retaspimycin was termi-nated because of toxicity (16). Hence, there is a need forsafer and possibly more effective HSP90 inhibitors to testin this disease.

Here,we report on the activity of thenon-geldanamycinHSP90 inhibitor, AT13387 (Fig. 1A), in GIST in vitro andin vivomodels. AT13387 is a potent small-moleculeHSP90inhibitor, which is currently being evaluated in the clinicin a phase II GIST trial (ClinicalTrials.gov Identifier:NCT01294202) in combination with imatinib (Fig. 1B). Itwas discovered using a fragment-based approach andhassignificant antitumor activity in a number of in vitro andin vivo cancer models (17, 18). Here, we show that thisinhibitor is active in both imatinib-sensitive and -resistantGIST models and that combination with imatinib in animatinib-resistant model enhances tumor growth inhibi-tion over either of the monotherapies. These data supportthe current clinical testing of AT13387 in this disease.

Materials and MethodsMaterials

AT13387 was synthesized at Astex Pharmaceuticalsand stored as a lyophilized powder. Synthesis of AT13387is asdescribedbyWoodheadandcolleagues (18). Imatinibmesylate and sunitinib malate were purchased fromSequoia Research Products Ltd. All other reagents werepurchased from Sigma unless otherwise stated.

Cell culture and reagentsThe human GIST cell lines, GIST882, GIST430, and

GIST48 described in (11) and GIST-T1 in (19) were used.GIST48B (20) and GIST430A were derived from GIST48and GIST430, respectively, grown in the presence of 500nmol/L 17-AAG. GIST882 and GIST430 were grown inRPMI-1640 and Iscove’s Modified Dulbecco’s Medium,respectively, both supplemented with 15% FBS. GIST48was grown in F10 medium supplemented with 15% FBS,3 mg/mL bovine pituitary extract, and MITOþ SerumExtender (BD Biosciences). Cells were maintained at 37�Cin a humidified atmosphere of 5% CO2. All cell culturereagents were purchased from Invitrogen unless other-wise stated. GIST882, GIST430, GIST48, GIST-T1,GIST48B, and GIST430A cell lines and GIST430 xenografttumors were validated by genomic PCR for the continuedpresence of known KIT mutations and by single-nucleo-tide polymorphism profiles (21–23). All lines were cul-tured in vitro for no more than 6 months after PCR andsingle-nucleotide polymorphismvalidations. Response ofGIST882, GIST48, andGIST430 to imatinibwasmonitoredregularly to ensure the cell line characteristicsdidnot alter.

Proliferation assaysViability studies were carried out using the CellTiter-

Glo luminescent assay (Promega) as described previously(11) or by using Alamar Blue (Invitrogen) as describedpreviously (24) but with slight modifications. Briefly, 105

cells were seeded in 200 mL of complete culture mediumper well into flat-bottomed 96-well plates 1 day before thedrug treatment. Cells were incubated with compound in0.1% (v/v) dimethyl sulfoxide (DMSO) for up to 7 daysbefore viability was assessed by using Alamar Blue.

Western blot samples and analysisCells were seeded into 6-well plates at 106 cells per well

and incubated overnight at 37�C followed by treatmentwith AT13387 or imatinib for the indicated time. Floatingand adherent cells were collected and prepared asdescribed previously (25). Samples were resolved bySDS-PAGE on a NuPage Novex (4%–12% Bis-Tris gel)system, transferred to nitrocellulose membranes, andincubatedwith primary antibodies specific for: KIT, phos-pho-KIT; AKT, phospho-AKT (p-AKT); cleaved PARP,(ClPARP); ERK, phospho-ERK (p-ERK); S6, phospho-S6(p-S6); HSP90 (Cell Signaling Technologies); actin(Abcam); or HSP70 (Enzo Life Sciences or Santa CruzTechnologies) followed by either infrared-dye-labeledanti-rabbit or anti-mouse antibodies (Licor Bioscience).Blots were scanned to detect infrared fluorescence on theOdyssey infrared imaging system (Licor Biotechnology)and, where relevant, signal intensity was determined.

Xenograft efficacy studiesThe GIST430 xenograft study was conducted in United

Kingdom. The care and treatment of experimental ani-mals were in accordance with the United Kingdom Coor-dinating Committee for Cancer Research guidelines (26)

A

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Figure 1. Chemical structures of AT13387 (A) and imatinib (B).

Smyth et al.

Mol Cancer Ther; 11(8) August 2012 Molecular Cancer Therapeutics1800




and with United Kingdom Animals (Scientific Proce-dures) Act 1986 (27). GIST430 xenografts were preparedby subcutaneously injecting 5 � 106 cells in 50 mL serum-freemediummixedwith 50 mLMatrigel (�10mg/mL, BDBiosciences) into the right flanks of BALB/c severe com-bined immunodeficient (SCID) mice (Harlan). Tumorburden was estimated from caliper measurements andby applying the formula for an ellipsoid.The GIST-PSW (human GIST, with KIT exon 11 harbor-

ing p.K558_G565delinsR mutation) study was conductedin Belgium as approved by the committee of animal ethicsof the Catholic University of Leuven, Belgium. GIST-PSWtumor pieces were grafted bilaterally into nu/nu NMRImice (Janvier Laboratories) as described previously (28).Tumor burdenwas estimated from calipermeasurementsand by applying the formula for a cuboid.Drug treatment of the mice was started when tumors

were well-established. GIST430 tumors developed inapproximately 3 weeks and animals bearing tumorsin the range of 92 and 303 mm3 were included in thestudy (N ¼ 8 per group). GIST-PSW tumors requiredapproximately 9 weeks to establish. Animals bearing 1or 2 tumors between 2 and 2,737 mm3 were divided into4 groups of 7 animals. All drug-treated animals harbored2 tumors (N¼ 14).Control groups included2animalswith2 tumors and 5 with only 1 tumor (N ¼ 9). AT13387 wasdissolved in 17.5% (w/v) hydroxypropyl-b-cyclodextrinand administered intraperitoneally at 70 mg/kg once aweek for 4 weeks except for the first dose in GIST-PSWstudy, which was 80 mg/kg. Imatinib was dissolved insterile water and given orally at 50 mg/kg (13) for 3or 4 weeks. Both AT13387 and imatinib were given at10 mL/kg. Control animals received 17.5% (w/v) 2-hydroxypropyl-b-cyclodextrin intraperitoneally.A statistically significant (one-way ANOVA with

Dunnett multiple comparison test or unpaired t testconducted on GraphPad Prism version 3.02) slowing

of the relative increase in xenograft volume or regres-sion of tumors compared with the control group wasused to characterize efficacy. Statistically significantdifferences were defined as P < 0.05. Tolerability wasestimated by monitoring body weight loss and survivalover the course of the study.

Analysis of pharmacodynamic markers in tumorsample

Tumor-bearing animals were sacrificed at various timepoints after single or multiple doses of AT13387 or ima-tinib. Tumors (typically 4 per treatment or time point)were snap-frozen in liquid nitrogen and analysis of phar-macodynamic markers in tumor samples was carried outas described previously (25, 29) with the same antibodiesused in vitro. Total and phospho-KIT levels in the sametumor lysates were also quantified using Phospho(Tyr721)/Total c-Kit Whole Cell Lysate Kit and SECTORPR 400 reader from Meso Scale Discovery (MSD) accord-ing to manufacturer’s instruction.

Histologic analysesTumor and liver samples were embedded in paraffin

and sections were stained with hematoxylin and eosin toaid histologic interpretation. Tumor sections were alsoimmunostained for KIT (polyclonal antibody, DAKOCorp.) as previously described (30).

ResultsAT13387 inhibits proliferation in GIST cell lines

AT13387 is a potent, novel HSP90 inhibitor thathas previously been shown to have antiproliferativeactivity in a wide range of different cell lines (17, 18).Here, AT13387 was tested against a range of GIST celllines with different activating and secondary resistancemutations (Table 1). AT13387 was shown to inhibit

Table 1. Cell lines investigated in this study and their response to inhibitors

Cell linePrimary KITmutation

SecondaryKITmutation

Otherfindings

Resistancepharmacology

IC50, nmol/L

AT13387 17-AAG Imatinib Sunitinib

GIST882a p.K642E 82 140 111 26GIST-T1b p.V560_Y578del 36 29 45 25GIST430a p.V560_L576del p.V654A Imatinib-resistant 34 110 46%I at

300 nmol/Lc37

GIST430Ab p.V560_L576del p.V654A NQO1-negative 17-AAG–resistant 75 >1,000 N/D N/DGIST48a p.V560D p.D820A Imatinib/

sunitinib-resistant55 97 >1,000 >10,000

GIST48Bb p.V560D p.D820A KIT-independent Imatinib/sunitinib/17-AAG-resistant

310 890 >1,000 >1,000

aCells were incubated with drugs for 7 days.bCells were incubated with drugs for 6 days.cForty-six percent inhibition at 300 nmol/L. See also Supplementary Fig. S1 for typical dose–response curve.

HSP90 Inhibitor AT13387 in GIST Models

www.aacrjournals.org Mol Cancer Ther; 11(8) August 2012 1801




proliferation, of all imatinib-sensitive (GIST-T1,GIST882), imatinib-resistant (GIST430), and multipleTKI-resistant (GIST48) lines with sub-100 nmol/Lpotencies (Table 1, Supplementary Fig. S1). The imati-nib-resistant GIST cells were derived from patientsprogressing on imatinib and harbored secondary KITmutations in addition to the primary activating muta-tions found in both sensitive and resistant lines (Table 1;ref. 11). In addition, AT13387 also inhibited prolifera-tion of 17-AAG–resistant cell lines, GIST430A andGIST48B (IC50 values of 75 and 310 nmol/L, respective-ly; ref. 20), which were derived by prolonged culturingof GIST430 and GIST48 cells in the presence of 500nmol/L of the HSP90 inhibitor, 17-AAG.

AT13387 inhibits KIT signaling in both imatinib-sensitive and -resistant cell lines

KIT is a client for HSP90 (10). The ability of AT13387 todegrade KIT and therefore inhibit KIT signaling wasinvestigated in 3 cell lines (GIST882, GIST430, andGIST48). Treatment of all 3 cell lines with increasingconcentrations of AT13387 depleted HSP90 client pro-teins, KIT and AKT, and caused significant inhibitionof KIT signaling at concentrations of AT13387 above75 nmol/L for GIST882 and GIST430 and above100 nmol/L for GIST48 (Fig. 2A), which correlates wellwith the IC50 values determined by the viability assay forthese cell lines (Table 1). A time course evaluatingAT13387 treatment for up to 72 hours (Fig. 2B) indicated

Actin

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Figure 2. Dose- and time-dependence of AT13387 treatmenton HSP90 clients and KIT signalingin GIST cell lines. GIST882,GIST430, and GIST48 cells weretreated with the indicatedconcentrations of AT13387 for 24hours (A) or 250 nmol/L of AT13387for 6 to 72 hours (B). Samples wererun by SDS-PAGE andimmunoblotted with the indicatedantibodies.

Smyth et al.





that effects of HSP90 inhibition such as HSP70 inductionandKIT depletion could be seen after 6 hours of treatmentand that these effects were sustained for 72 hours bywhich time significant cell death was observed. Similareffects were seen in all 3 cell lines tested.To compare the effects of AT13387 treatment with

imatinib treatment in the 3 cell lines, cells were treatedwith varying concentrations of AT13387 or imatinib for24 hours (Fig. 3). As previously, AT13387 treatmentinduced HSP70, an indicator of HSP90 inhibition, in all3 cell lines with a concomitant depletion in the levels ofthe clients KIT, AKT, and their phospho-forms. Levels ofthe downstream signaling molecules, phospho-ERK andphospho-S6, were also decreased, indicating that KITsignaling was significantly inhibited in all cell lines. Incontrast, imatinib significantly inhibited KIT signaling,only in the imatinib-sensitive cell line, GIST882, with littlechange in the levels of phospho-KIT, phospho-AKT, andphospho-S6 seen in the imatinib-resistant cell lines,GIST430 andGIST48. An increase in cleaved PARP, indic-ative of apoptosis, was seen in all cells treated withAT13387, whereas again for cells treated with imatinib,an increase in cleaved PARP was only seen for the ima-tinib-sensitive cell line. These data confirm that inhibitionof HSP90 leads to inhibition of KIT signaling pathways inGIST cell lines driven by activating mutations in KIT andthat this inhibition is irrespective of secondary mutationsin KIT, which can render cells resistant to imatinib.

Characterization of the novel imatinib-resistantGIST430 xenograft modelTo date, only 2 imatinib-resistant GIST xenograft mod-

els have been reported, GIST48 and GIST-BOE (13).Because the GIST430 cell line showed resistance toimatinib in vitro (11), this cell line was a good candidatefor establishing a novel imatinib-resistant GIST xenograftmodel.

GIST430 cells harbor a heterozygous primary mutationin exon 11 (p.V560_L576del) and a secondary resistancemutation in exon 13 (V654A). This cell line failed todevelop subcutaneous solid tumors in nude mice with orwithout Matrigel, or in SCID mice without Matrigel (datanot shown). However, when 5 � 106 GIST430 cells weremixed with Matrigel then injected into SCID mice, solidtumorsdeveloped inmore than90%of the animals after 19to 22 days. In 3 independent experiments, the averagesizes of all tumors at this time were 146 to 184 mm3.Tumors that were selected for efficacy studies were in therange of 92 and 303mm3 (57%–60% of the total number ofanimals inoculated). The vehicle-treated tumors grewexponentially with a doubling time of 5.3 to 7.0 days.

Total KIT expression in tumors was confirmed byWestern blotting (Figs. 4 and 5) and immunohistochem-istry, in which uniform total KIT staining was observed(data not shown). Presence of phosphorylated KIT and itsdownstream signaling through AKT, ERK, and S6 werealso confirmed by Western blotting (Figs. 4 and 5).

Sensitivity of the GIST430 xenograft to imatinib wastested by orally dosing the animals with imatinib at 50mg/kg twice a day as described later.

AT13387 significantly inhibits growth of bothimatinib-sensitive and -resistant xenograft modelsin vivo

The antitumor effects of AT13387 were tested at itsmaximum-tolerated dose of 70 mg/kg in a once-weeklydose schedule as a single agent as well as in combinationwith imatinib in both imatinib-sensitive (GIST-PSW) andimatinib-resistant (GIST430) xenograft models (Fig. 6). TheGIST430model has been described earlier. GIST-PSW is animatinib-sensitive model with a primary KIT mutation inexon 11 (with p.K558_G656delinsR mutation; ref. 28).

In both xenograft models, AT13387 was given intraper-itoneally at 70 mg/kg once a week. AT13387 inhibited

Figure 3. Effects of AT13387 andimatinib on KIT signaling in imatinib-sensitive and -resistant GIST celllines. GIST882 (A), GIST430 (B), andGIST48 (C) cells were incubated withthe indicated concentrations ofAT13387or imatinib (IM) for 24 hours.Sampleswere runbySDS-PAGEandimmunoblotted with the indicatedantibodies. This study was typical ofat least 3 experiments.

ClPARP

GIST882 GIST430 GIST48

0.510.250.510 0.25 µmol/L

AT13387 IM

0.510.250.510 0.25 µmol/L

AT13387 IM

0.510.250.510 0.25 µmol/L

AT13387 IM

p-KITY703

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tumor growth in bothmodels (P< 0.001 against the vehiclecontrol; Fig. 6A and B). As expected, twice-daily oraltreatment with imatinib at 50 mg/kg caused regressionof the imatinib-sensitive GIST-PSW tumors during the 22-day treatment period, but not of the imatinib-resistantGIST430 tumors. Imatinib caused moderate but statisti-cally significant reduction in GIST430 tumor growth (P <0.001), although growth inhibition was not as marked asthat caused by AT13387 treatment. However, combiningAT13387 and imatinib enhanced tumor growth inhibitionover the maximal effect of either of the monotherapies inthe GIST430 xenograft.

Single-agent treatment with AT13387 caused meanbody weight loss of 10% after the first dosing in SCIDmice but this effect was transient, lasting no more than 6days (Fig. 6C). Less body weight loss was observed onsubsequent dosing. No significant tolerability issues wereassociated with imatinib monotherapy. Importantly, thecombination of AT13387 and imatinib resulted in noincreased toxicity in mice with either GIST-PSW orGIST430 tumors (Fig. 6C). In addition, at the end of theGIST430 xenograft study, livers of the animals were ana-lyzed by hematoxylin and eosin staining to assess if any ofthe treatments had caused liver damage, such as thosereported for IPI-493 (13). No histologic differences wereobserved between the animals in the 4 different treatmentgroups (data not shown), indicating none of the treat-ments had caused significant histologic hepatotoxicity.

KIT signaling is inhibited by AT13387 in xenografttissues

The effect of AT13387 treatment on KIT signaling inxenograft tissue was investigated in both the GIST-PSWandGIST430 xenografts (Fig. 4). Analyses of protein levels

by Western blotting in the xenograft tumors from micetreated with a single 70 mg/kg dose of AT13387 showedsimilar effects onKIT signaling to those seen in cell lines.Areduction in the phospho-forms of the client proteins, KITand AKT, was seen in parallel with inhibition of down-stream signaling through ERK and S6 for at least 24 hoursin both models (Fig. 4). HSP70, an indicator of HSP90inhibition, was induced over the same time period.This confirms that AT13387 brings about ablation of KITsignaling, as a result ofHSP90 inhibition, in both imatinib-sensitive and -resistant xenograftswith variousKITmuta-tions and suggests that downregulation of signaling forthis length of time is sufficient to trigger apoptosis andinhibition of tumor growth.

AT13387 treatment was also compared with imatinibas monotherapy and to the AT13387 and imatinib com-bination. As expected, imatinib treatment depleted phos-pho-KIT levels in the GIST-PSW tumors at 4 hours afterthe last dose on day 22, similarly to AT13387 treatment(Supplementary Fig. S2). In contrast, imatinib caused littleeffect on phospho-KIT and its signaling in GIST430tumors (Fig. 5), confirming reduced sensitivity of thesetumors to imatinib. In both models, the combination ofAT13387 and imatinib caused a similar depletion of phos-pho-KIT and inhibition of downstream signaling to thatseen with AT13387 alone (Fig. 5, Supplementary Fig. S2).

DiscussionPrevious experiments in cell lines and in vivo models

have suggested that HSP90 inhibition might be an effec-tive treatment option for GIST (10–14). GIST subcloneswith various resistance mutations are selected duringtreatment with the currently available therapies, imatinib

72 h48 h24 h12 h6 h

GIST-PSW (IM-sensitive)

UntreatedUntreated 72 h48 h24 h12 h6 h

GIST430 (IM-resistant)

p-KITY703

KIT

p-AKTS473

AKT

HSP70

p-ERKT202/Y204

ERK

p-S6S240/244

S6

Actin

Figure 4. Duration of effects ofAT13387 treatment on KITsignaling in imatinib (IM)-sensitiveand -resistant GIST xenografts.Mice bearing GIST-PSW orGIST430 xenograft tumors weretreated with AT13387 once at 70mg/kg. Animals were sacrificed atthe indicated time points andprotein expression in tumors wasevaluatedbyWestern blotting. Tworandom tumors out of 4 for eachtime point are shown.

Smyth et al.





and sunitinib, and these can be heterogeneous within thesame patient at different metastatic sites and even withinthe same tumor nodule (7). Imatinib-resistant GISTremains largely KIT-dependent and so targeting KIT viainhibition of its chaperone, HSP90, which is essential forstability of all mutant KIT proteins, is particularlyattractive.AT13387 is a novel, small-molecule inhibitor of HSP90

currently being evaluated in combination with imatinib,in a phase II GIST trial (ClinicalTrials.gov Identifier:NCT01294202). Here, we have shown that AT13387 iseffective at inhibiting proliferation and KIT signaling, via

the depletion of KIT, in both imatinib-sensitive and -resis-tant GIST cell lines in vitro. In addition, we have shownthat AT13387 has significant antitumor activity in bothimatinib-sensitive and -resistant xenograft models. Thisconfirms that HSP90 inhibition is a viable treatmentoption for KIT-driven GIST regardless of secondary ima-tinib resistance mutations and highlights AT13387 as anexcellent candidate for clinical use in this disease.

Inter- and intralesional heterogeneity of resistancemutations often coexist in the same patient (7). In suchcases only some lesionsmay respond to an individual TKIwhereas others remain resistant. AT13387 was effective

Figure 5. Comparison of the effectsof AT13387 and imatinib dosedindividually or in combination on KITsignaling in xenograft tumors. Micebearing GIST430 xenograft tumorswere treated with AT13387 once at70 mg/kg, imatinib (IM) twice at 50mg/kg, or the combination of 2treatments. This represents the day 2of the efficacy studies. Animals weresacrificed at 24 hours after the firstdose and tumors were evaluated byWestern blotting (A). Total andphospho-KIT in the same tumorlysates were also analyzed by MSD(B). All data points represent themean � SE of 4 tumors from 4individual animals. �, P < 0.05 versuscontrol.

CombinationIMAT13387Untreated

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against both the GIST430 and GIST48 cell lines, whichhave different resistance-inducing KITmutations (V654AandD820A, respectively) confirming that effective HSP90inhibition may have a distinct advantage over furtherTKIs for treatment in GIST once resistance has arisen. Inaddition to multiple resistant clones, imatinib-sensitiveand -resistant clones can also coexist in the same patient

(7, 11). A combination of AT13387 and imatinib thereforecould provide benefit in targeting this heterogeneity,with imatinib active on the sensitive clones and AT13387on those that are either sensitive or resistant. In ourstudies, AT13387 was well tolerated in combination withimatinib and in the imatinib-resistant GIST430 model thecombination had improved efficacy over either of themonotherapies, supporting the idea of combining thesetherapies in the clinic. So far clinical trials have failed toprove that drug combinations achieve better results than asingle agent TKI in GIST, but our preclinical data suggestthat the combination of theHSP90 inhibitor, AT13387, andimatinib may be more successful.

Interestingly, AT13387 also still inhibited proliferationof cell lines that had developed resistance to 17-AAG,indicating that the resistance mechanism to the geldana-mycin analogues is not universal to all HSP90 inhibitorsand can be circumvented by use of an alternative struc-tural class.

Previously, the related geldanamycins, 17-AAG, retas-pimycin and IPI-493 have been tested in GIST models(11, 13, 14), but this class of inhibitors has shown limita-tions in the clinic due to formulation issues, namelyadverse effects of Cremophor (31) or DMSO (32) includedin the formulation of poorly soluble 17-AAG and hepa-totoxicity (15, 33). A retaspimycin phase III GIST trial wasterminated early due to a number of toxicity issues,including liver failure that possibly led to excessmortalityin one trial (16), and recent preclinical data on this drugshowed liver damage in mice when combining withimatinib (14). Hence, there is a need for new compoundswith amore favorable pharmacologic profile for testing inGIST and particularly for compounds that can be safelyadministered in combination with the first-line therapy,imatinib. AT13387 appears to be an excellent candidate asits combination with imatinib was well tolerated in micewith no apparent liver damage.

GIST in vivo models have proven difficult to establishand only 2 imatinib-resistant models have been prev-iously described: GIST48, which has a secondary exon17 resistancemutation, andGIST-BOE,whichhas a single-copy KIT with a mutation in exon 9 and exhibits relative(dose-dependent) resistance to imatinib (13, 28). Bothmodels are slow growing and have higher success rateswhen serially transplanted. In contrast, the GIST430 xeno-graft described here can be more readily established byinjecting cells cultured in vitro and the resulting tumorshave a faster growth rate. The response of our GIST430xenograft model to imatinib was markedly different tothe regressions seen in the imatinib-sensitive GIST-PSWmodel. This confirms a level of imatinib resistancealthough a moderate response, rather than completeinsensitivity, was observed. This result is consistent withour in vitro data with this cell line, where inhibition ofproliferation can be observed at high concentrations ofimatinib (Supplementary Fig. S1). Similarly, the previous-ly described GIST-BOE and GIST48 xenografts were onlypartially resistant to imatinib, with treatment still

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Figure 6. Antitumor activities of AT13387 and imatinib as single agentsand in combination against imatinib-sensitive and -resistant GISTxenografts. Mice bearing GIST-PSW (A) and GIST430 (B) xenografttumors were treated with AT13387 once a week intraperitoneally at 70mg/kg (~), imatinib (IM) twice a day orally at 50 mg/kg (!), or acombination of AT13387 and imatinib (*). Control tumor-bearinganimals received vehicle (&). Relative tumor volumes (RTV) are shown.Bodyweight changes in theGIST430study are shown inC.All data pointsare mean � SE of at least 7 tumors per animals. GIST430 data arerepresentative of 2 studies.

Smyth et al.





resulting in stabilization of the xenograft tumors (13). Theavailability of another GIST xenograft model, with differ-ent secondary resistance mutations to the previouslyestablished GIST48, adds to the tools available for thefuture evaluation of potential GIST-active compounds.GIST is not the only cancerwhereKIT activation is a key

driver of disease (34). The majority of cases of adultsystemic mastocytosis is associated with the D816V KITgain-of-function mutation (35), whereas a proportion ofcore binding factor acute myeloid leukemias also havemutations at the 816 position or in exon 8 (36, 37). TheD816Vmutation, positioned in the kinase activation loop,causes KIT activation and confers resistance to imatinib(38).KIThas also been shown tobe a therapeutic target in asubset ofmelanomaswhereKIT-activatingmutations andoverexpression have been observed (39, 40). Hence,HSP90 inhibitors such as AT13387 could have furtheruses in KIT-driven disease.Overall, the effectiveness of AT13387 against both ima-

tinib-sensitive and -resistant GIST and the fact that it iswell tolerated in mice in combination with the first-linetherapy, imatinib, suggest it is a promising candidate foruse in this disease. These data as awhole strongly supportthe current testing of AT13387 in combination with ima-tinib in GIST clinical studies and validate the novel mech-anismof combining anHSP90 inhibitorwithTKIs inGIST.

Disclosure of Potential Conflicts of InterestT. Smyth, J.E. Curry, A.M. Rodriguez-Lopez, J. Lyons, N.T. Thompson,

and N.G. Wallis are employees of Astex Pharmaceuticals. T.V. Looy, A.Wozniak, and P. Sch€offski in Leuven, Belgium received research supportfromAstex Pharmaceuticals. P. Sch€offski has also received research grantsfor GIST translational work from Novartis Pharmaceuticals and Infinity

Pharmaceuticals. J.F. Lyons has a relationshipwithOnyxPharmaceuticals.A. Wozniak received honoraria from Novartis. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: T. Smyth, T.V. Looy, J.E. Curry, A. Wozniak, J.A.Fletcher, P. Schoffski, J.F. Lyons, N.T. Thompson, N.G. WallisDevelopment of methodology: T. Smyth, J.E. Curry, A.M. Rodriguez-Lopez, J.G. Morgan, M.M. Mayeda, P. SchoffskiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Smyth, T.V. Looy, J.E. Curry, A. Wozniak,M. Zhu, M.M. Mayeda, J.A. Fletcher, P. SchoffskiAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):T. Smyth, T.V. Looy, J.E. Curry,A.Wozniak,M. Zhu, M.M. Mayeda, J.A. Fletcher, P. Schoffski, N.T. Thompson, N.G.WallisWriting, review, and/or revision of the manuscript: T. Smyth, T.V. Looy,J.E. Curry, A.Wozniak, J.A. Fletcher, P. Schoffski, J.F. Lyons, N.T. Thomp-son, N.G. WallisAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T. Smyth, J.E. Curry, P. SchoffskiStudy supervision: J.E. Curry, J.F. LyonsDevelopment of GIST cell lines: R. Donsky

AcknowledgmentsThe authors thank Rachel McMenamin, Jon Lewis, and Brent Graham

for their help with cell culture, in vivowork, and scientific discussion andMaria Debiec-Rychter, who has supported the xenograft work in Leuven,Belgium.

Grant SupportJ.A. Fletcher is supported in part by funding from GI SPORE

1P50CA127003, the Life Raft Group, the GIST Cancer Research Fund, andthe Virginia and Daniel K. Ludwig Trust for Cancer Research.

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 December 22, 2011; revised April 3, 2012; accepted April 20,2012; published OnlineFirst June 19, 2012.

References1. Antonescu CR. The GIST paradigm: lessons for other kinase-driven

cancers. J Pathol 2011;223:251–61.2. Hornick JL, Fletcher CD. The role of KIT in themanagement of patients

with gastrointestinal stromal tumors. Hum Pathol 2007;38:679–87.3. Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S ,

et al. Gain-of-function mutations of c-kit in human gastrointestinalstromal tumors. Science 1998;279:577–80.

4. Debiec-Rychter M, Dumez H, Judson I, Wasag B, Verweij J, BrownM ,et al. Use of c-KIT/PDGFRA mutational analysis to predict the clinicalresponse to imatinib in patientswith advanced gastrointestinal stromaltumours entered on phase I and II studies of the EORTC Soft Tissueand Bone Sarcoma Group. Eur J Cancer 2004;40:689–95.

5. Heinrich MC, Corless CL, Demetri GD, Blanke CD, von MM, JoensuuH, et al. Kinase mutations and imatinib response in patients withmetastatic gastrointestinal stromal tumor. J Clin Oncol 2003;21:4342–9.

6. Gramza AW, Corless CL, Heinrich MC. Resistance to tyrosine kinaseinhibitors in gastrointestinal stromal tumors. Clin Cancer Res 2009;15:7510–8.

7. Liegl B, Kepten I, Le C, Zhu M, Demetri GD, Heinrich MC , et al.Heterogeneity of kinase inhibitor resistance mechanisms in GIST.J Pathol 2008;216:64–74.

8. Csermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinicalapplications. A comprehensive review. Pharmacol Ther 1998;79:129–68.

9. Solit DB, Rosen N. Hsp90: a novel target for cancer therapy. Curr TopMed Chem 2006;6:1205–14.

10. Fumo G, Akin C, Metcalfe DD, Neckers L. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is effective in down-regulatingmutated, constitutively activated KIT protein in human mast cells.Blood 2004;103:1078–84.

11. Bauer S, Yu LK, Demetri GD, Fletcher JA. Heat shock protein 90inhibition in imatinib-resistant gastrointestinal stromal tumor. CancerRes 2006;66:9153–61.

12. Dewaele B, Wasag B, Cools J, Sciot R, Prenen H, Vandenberghe P ,et al. Activity of dasatinib, a dual SRC/ABL kinase inhibitor, and IPI-504, a heat shock protein 90 inhibitor, against gastrointestinal stromaltumor-associatedPDGFRAD842Vmutation. ClinCancerRes 2008;14:5749–58.

13. Floris G, Sciot R, Wozniak A, Van LT,Wellens J, Faa G , et al. The novelheat shock protein 90 inhibitor, IPI-493, is highly effective in humangastrostrointestinal stromal tumor (GIST) xenografts carrying hetero-geneous KIT mutations. Clin Cancer Res 2011;17:5604–14.

14. Floris G, Debiec-Rychter M, Wozniak A, Stefan C, Normant E, Faa G ,et al. The heat shock protein 90 inhibitor IPI-504 induces KIT degra-dation, tumor shrinkage and cell proliferation arrest in xenograftsmodels of gastrointestinal stromal tumors. Mol Cancer Ther 2011;10:1897–908.

15. BiamonteMA, Van deWater R, Arndt JW, Scannevin RH, Perret D, LeeWC. Heat shock protein 90: inhibitors in clinical trials. J Med Chem2010;53:3–17.

16. Demetri GD, Le Cesne A, Von Mehren M, Chmielowski B, Bauer S,Chow WA, et al.Final results from a phase III study of IPI-504 (retas-pimycin hydrochloride) versus placebo in patients (pts) with gastroin-testinal stromal tumors (GIST) following failure of kinase inhibitor






therapies [abstract]. 2010 In: Proceedings of the GastrointestinalCancers Symposium; 2010 Jan 22. Alexandria, VA: American Societyof Clinical Oncology.

17. Graham B, Curry J, Smyth T, Fazal L, Feltell R, Harada I , et al. TheHSP90 inhibitor, AT13387, displays a long duration of action in vitroand in vivo in non-small cell lung cancer. Cancer Sci 2011;103:522–7.

18. WoodheadAJ,AngoveH,CarrMG,ChessariG,CongreveM,CoyleJE ,et al. Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4-methylpi-perazin-1-ylmethyl)-1,3-dihydroisoindol-2-yl]methanone (AT13387), anovel inhibitor of the molecular chaperone Hsp90 by fragment baseddrug design. J Med Chem 2010;53:5956–69.

19. Taguchi T, Sonobe H, Toyonaga S, Yamasaki I, Shuin T, Takano A ,et al. Conventional and molecular cytogenetic characterization of anew human cell line, GIST-T1, established from gastrointestinal stro-mal tumor. Lab Invest 2002;82:663–5.

20. GunaratnamM, SwankS, Haider SM,GalesaK, ReszkaAP, BeltranM ,et al. Targeting human gastrointestinal stromal tumor cells with aquadruplex-binding small molecule. J Med Chem 2009;52:3774–83.

21. HeinrichMC, Corless CL, BlankeCD, Demetri GD, JoensuuH, RobertsPJ , et al. Molecular correlates of imatinib resistance in gastrointestinalstromal tumors. J Clin Oncol 2006;24:4764–74.

22. Lassus H, Sihto H, Leminen A, Nordling S, Joensuu H, Nupponen NN ,et al. Genetic alterations and protein expression of KIT and PDGFRA inserous ovarian carcinoma. Br J Cancer 2004;91:2048–55.

23. McLean SR, Gana-Weisz M, Hartzoulakis B, Frow R, Whelan J, Sel-wood D , et al. Imatinib binding and cKIT inhibition is abrogated by thecKIT kinase domain I missense mutation Val654Ala. Mol Cancer Ther2005;4:2005–15.

24. Squires MS, Feltell RE, Wallis NG, Lewis EJ, Smith DM, Cross DM ,et al. Biological characterization of AT7519, a small-molecule inhibitorof cyclin-dependent kinases, in human tumor cell lines. Mol CancerTher 2009;8:324–32.

25. Curry J, Angove H, Fazal L, Lyons J, Reule M, Thompson N , et al.AuroraBkinase inhibition inmitosis: strategies for optimising theuseofaurora kinase inhibitors such as AT9283. Cell Cycle 2009;8:1921–9.

26. WorkmanP, Aboagye EO, Balkwill F, Balmain A, BruderG, Chaplin DJ ,et al. Guidelines for the welfare and use of animals in cancer research.Br J Cancer 2010;102:1555–77.

27. Hollands C. The Animals (scientific procedures) Act 1986. Lancet1986;2:32–3.

28. Floris G, Debiec-Rychter M, Sciot R, Stefan C, Fieuws S, Machiels K ,et al. High efficacy of panobinostat towards human gastrointestinalstromal tumors in a xenograft mouse model. Clin Cancer Res2009;15:4066–76.

29. Banerji U, Walton M, Raynaud F, Grimshaw R, Kelland L, Valenti M ,et al. Pharmacokinetic-pharmacodynamic relationships for the heatshock protein 90 molecular chaperone inhibitor 17-allylamino, 17-demethoxygeldanamycin in human ovarian cancer xenograft models.Clin Cancer Res 2005;11:7023–32.

30. Penner CR, Folpe AL, Budnick SD. C-kit expression distinguishessalivary gland adenoid cystic carcinoma from polymorphous low-grade adenocarcinoma. Mod Pathol 2002;15:687–91.

31. Modi S, Stopeck AT, Gordon MS, Mendelson D, Solit DB, Bagatell R ,et al. Combination of trastuzumab and tanespimycin (17-AAG, KOS-953) is safe and active in trastuzumab-refractory HER-2 overexpres-sing breast cancer: a phase I dose-escalation study. J Clin Oncol2007;25:5410–7.

32. Solit DB, Osman I, Polsky D, Panageas KS, Daud A, Goydos JS , et al.Phase II trial of 17-allylamino-17-demethoxygeldanamycin in patientswith metastatic melanoma. Clin Cancer Res 2008;14:8302–7.

33. Solit DB, Ivy SP, Kopil C, Sikorski R,MorrisMJ, Slovin SF , et al. Phase Itrial of 17-allylamino-17-demethoxygeldanamycin in patients withadvanced cancer. Clin Cancer Res 2007;13:1775–82.

34. Heinrich MC, Blanke CD, Druker BJ, Corless CL. Inhibition of KITtyrosine kinase activity: a novelmolecular approach to the treatment ofKIT-positive malignancies. J Clin Oncol 2002;20:1692–703.

35. Lim KH, Tefferi A, Lasho TL, Finke C, Patnaik M, Butterfield JH , et al.Systemicmastocytosis in 342 consecutive adults: survival studies andprognostic factors. Blood 2009;113:5727–36.

36. Paschka P, Marcucci G, Ruppert AS, Mrozek K, Chen H, Kittles RA ,et al. Adverse prognostic significance of KIT mutations in adult acutemyeloid leukemia with inv(16) and t(8;21): a Cancer and LeukemiaGroup B study. J Clin Oncol 2006;24:3904–11.

37. Schnittger S, Kohl TM, Haferlach T, Kern W, Hiddemann W, Spieker-mann K , et al. KIT-D816 mutations in AML1-ETO-positive AML areassociated with impaired event-free and overall survival. Blood2006;107:1791–9.

38. MaY, Zeng S, Metcalfe DD, Akin C, Dimitrijevic S, Butterfield JH , et al.The c-KITmutation causing humanmastocytosis is resistant toSTI571and other KIT kinase inhibitors; kinases with enzymatic site mutationsshow different inhibitor sensitivity profiles than wild-type kinases andthose with regulatory-type mutations. Blood 2002;99:1741–4.

39. Went PT, Dirnhofer S, Bundi M,MirlacherM, Schraml P, Mangialaio S ,et al. Prevalence of KIT expression in human tumors. J Clin Oncol2004;22:4514–22.

40. Woodman SE, Davies MA. Targeting KIT in melanoma: a paradigm ofmolecular medicine and targeted therapeutics. Biochem Pharmacol2010;80:568–74.

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2012;11:1799-1808. Published OnlineFirst June 19, 2012.Mol Cancer Ther Tomoko Smyth, Thomas Van Looy, Jayne E. Curry, et al. ModelsImatinib-Sensitive and -Resistant Gastrointestinal Stromal Tumor The HSP90 Inhibitor, AT13387, Is Effective against

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