Cancer Therapy: Preclinical Activity of Dasatinib, a Dual SRC/ABL Kinase Inhibitor, and IPI-504, aHeatShockProtein90Inhibitor,againstGastrointestinal Stromal Tumor ^ Associated PDGFRAD842V Mutation Barbara Dewaele,1BartoszWasag,1Jan Cools,1,4 Raf Sciot,2 Hans Prenen,3 PeterVandenberghe,1 AgnieszkaWozniak,3 Patrick Scho« ffski,3 Peter Marynen,1,4 and Maria Debiec-Rychter1 Abstract Purpose: Activating mutations in platelet-derived growthfactor receptor- a (PDGFRA) have been reported in f5% to 10% of patients withgastrointestinal stromal tumors (GIST). Imatinib efficiently inhibits the juxtamembrane PDGFRA mutations, whereas many tyrosine kinase domain activation loop PDGFRA mutations confer primary resistance to imatinib. In this study, we compared the efficacy of second-line tyrosine kinase inhibitors such as dasatinib, sorafenib, and nilotinib against two GIST-related PDGFRA mutants, PDGFRAD842V and PDGFRADDIM842-844. In addition, we sought to investigate the inhibitory effect of the heat shock protein 90 inhibitor, IPI-504, on these mutants. Experimental Design: Primary imatinib-resistant tumor cells and cell lines expressing imatinib- resistant PDGFRAD842V or imatinib-sensitive PDGFRADDIM842-844 mutants were treated with different concentrations of dasatinib, sorafenib, nilotinib, and IPI-504. The effect of treatment on proliferation, survival, and signaling was determined. Results: All inhibitors tested exhibited a high efficacy toward the PDGFRADDIM842-844 mutant. In contrast, ex vivo and in vitro assays revealed that only dasatinib potently inhibited the D842V PDGFRA isoform withan IC 50 value of 62 nmol/L. Sorafenib and nilotinib were significantly less efficacious against this mutation, inhibiting the PDGFRA kinase activity at >1, 0 0 0 a nd >5,000 nmol/L, and suppressing the proliferation of the cells expressing the PDGFRAD842V mutant withan IC 50 value of 239 and 1,310 nmol/L, respectively. IPI-504 treatment potently inhibited PDGFRA kinase activity by inducing the degradation of PDGFRAD842V and PDGFRADDIM842-844 at 256 and 182 nmol/L, respectively. Conclusions: Treatment with dasatinib or the heat shock protein 90 inhibitor IPI-504 may provide a therapeutic alternative for GIST patients whose tumors carry the imatinib-resistant PDGFRAD842V mutant isoform. Gastrointestinal stromal tumors (GIST) comprise the largest tyrosine kinase homologous to KIT (5). Activating KIT and subset of mesenchymal tumors that develop along the PDGFRA mutations in GISTs differ in type and affect different gastrointestinal tract (1, 2). Constitutive activation of the KIT receptor domains (6). Therapeutic inhibition of KIT or tyrosine kinase through oncogenic mutations is an early and PDGFRA by the small-molecule inhibitor imatinib gives a probably initiating event in the majority of GISTs (3, 4). clinical response in the majority of advanced GISTs (3). Alternatively, a subset of GISTs express a constitutively activated However, early resistance has been reported in 10% to 20% platelet-derived growth factor receptor-a (PDGFRA) mutant, a of patients. It is well known that sensitivity to imatinib depends on the location of the KIT and PDGFRA gene mutation (3). Based on in vitro studies and subsequent clinical trials, it has been shown that a subset of GISTs harboring KIT and PDGFRA 1 2 3 Authors’Affiliations: Departments of Human Genetics, Pathology, and General mutations in the second tyrosine kinase domain (activation Medical Oncology, K.U. Leuven, and 4VIB Department of Molecular and Developmental Genetics,VIB, Leuven, Belgium loop domain) do not respond well to imatinib treatment Received 2/27/08; revised 4/28/08; accepted 5/22/08. (3, 7–10). The oncogenic PDGFRA mutations detected in Grant support: Life Raft Group, the Fonds voor Wetenschappelijk Onderzoek GISTs mainly involve exons 12, 14, or 18, and account for Vlaanderen (G.0286.05; M. Debiec-Rychter), and by a Concerted Action grant f5% to 10% of all GIST mutations. Wild-type PDGFRA is 2006/14 from the K.U. Leuven. sensitive to imatinib (11), but only a portion of the consti- The costs of publication of this article were defrayed in part by the payment of page advertisement tutively activated PDGFRA isoforms identified in GISTs are charges. This article must therefore be hereby marked in accordance D842V with18 U.S.C. Section 1734 solely to indicate this fact. potently inhibited by imatinib (3). The missense PDGFRA Requests for reprints: Maria Debiec-Rychter, Department of Human Genetics, mutation resulting in the substitution of aspartic acid to valine University of Leuven, O&N Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. at codon 842 accounts for f60% of all PDGFRA mutations Phone: 32-16-347218; Fax: 32-16-346210; E-mail: Maria.Debiec-Rychter@ med.kuleuven.be. known in GISTs (5). It confers primary resistance to imatinib F 2008 American Association for Cancer Research. in vitro and in vivo (3, 5, 8, 12), resistance to sunitinib in vitro doi:10.1158/1078-0432.CCR-08-0533 (13), and resistance to nilotinib in vitro and in vivo (14). www.aacrjournals.org 5749 Clin Cancer Res 2008;14(18) September 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical Conversely, we have previously shown that PKC412 efficiently oncoprotein degradation, but the effect of HSP90 inhibitors on inhibited the PDGFRAD842V mutant at the concentration of GISTs bearing PDGFRA mutations has thus far not been tested. 1 Amol/L (12). In this report, we examined the efficacy of second-generation Because resistance of GIST patients to imatinib is a tyrosine kinase inhibitors nilotinib, dasatinib, and sorafenib for D continuous clinical challenge, multiple novel therapeutic the inhibition of PDGFRAD842V-expressing and PDGFRA DIM842-844– strategies are under development. Novel small molecule expressing cells. In addition, we tested the potency of the HSP90 compounds being tested in clinical trials include nilotinib, inhibitor, IPI-504, to induce PDGFRA oncoprotein degradation sorafenib, and dasatinib. as a therapeutic alternative for imatinib-resistant, PDGFRA mutant In addition, heat shock protein 90 (HSP90) inhibition, GISTs. causing degradation of wild-type and imatinib-resistant KIT mutant proteins, has recently been validated in preclinical Materials and Methods studies as an alternative therapeutic strategy to challenge the heterogeneity of KIT imatinib-resistant mutations in patients Inhibitors. The inhibitors, imatinib mesylate (Glivec/Gleevec, with GIST (15). The PDGFRA protein is also subject to HSP90- Novartis), sorafenib (BAY-43-9006, Bayer AG), nilotinib (AMN107, mediated protection from proteasomal degradation in cancer Novartis), dasatinib (BMS-354825, Bristol-Meyers-Squibb), and IPI-504 cells (16) and HSP90 inhibition may cause effective PDGFRA were provided by Infinity Pharmaceuticals, Inc. and MedImmune Inc. Fig. 1. In vitro effect of imatinib, nilotinib, sorafenib, and dasatinib on PDGFRADDIM842-844 ^expressingandPDGFRAD842V-expressing Ba/F3 cells. The dose-response proliferation curves of PDGFRADDIM842-844 ^ transduced (A)andPDGFRAD842V-transduced (B) Ba/F3 cells, treated withimatinib, nilotinib, sorafenib, and dasatinib for 48 h. Points, the average results of two experiments done in triplicate plotted with the curve-fitting Origin software; bars, SD.The calculated IC50 for eachinhibitor is indicated. Representative results from apoptotic assays of PDGFRADDIM842-844 ^ transduced (C)andPDGFRAD842V-transduced (D) Ba/F3 cells. All compounds induced significant apoptosisbetween100and500nmol/LincellsexpressingPDGFRADDIM842-844. In contrast, only dasatinib showed significant induction of apoptosis at 500 nmol/L in cells expressing PDGFRAD842V. Clin Cancer Res 2008;14(18) September 15, 2008 5750 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Therapeutic Alternative for PDGFRAD842V Mutant Table 1. Comparison of in vitro apoptosis induction of imatinib-sensitive PDGFRADDIM842-844 and imatinib- resistant PDGFRAD842V mutants expressed in the Ba/F3 cells PDGFRA#DIM842-844 PDGFRAD842V Normal (%) Necrotic (%) Apoptotic (%) Normal (%) Necrotic (%) Apoptotic (%) Vehicle 85.1 9 5.7 84.2 7.6 7.4 Imatinib 0.1 Amol/L 71.8 15.1 12.9 85.9 6.8 6.6 0.5 Amol/L 60.9 20.8 18.1 87.7 4.7 7.3 Nilotinib 0.1 Amol/L 85 7.8 7.1 87.7 6.9 4.5 0.5 Amol/L 73.8 13.4 12.5 88.4 5.7 5.2 Sorafenib 0.1 Amol/L 74.1 14.1 11.5 87.9 6.6 5 0.5 Amol/L 66.5 16.7 16.8 89.4 5.9 4.5 Dasatinib 0.1 Amol/L 46.1 26.6 27 77.9 13.3 8.3 0.5 Amol/L 39.3 30.3 28.9 59.4 24.1 15.4 IPI-504 0.1 Amol/L 73.1 14.6 12.1 80.3 11.9 6.5 0.5 Amol/L 48.4 28.6 22.1 37.8 37.8 22.8 A 10 mmol/L stock solution of the inhibitors, dissolved in DMSO or a citric salt buffer for IPI-504) and incubated for 2 h at +37jC. After a citric salt buffer (for IPI-504) was stored at -80jC. The inhibitors were wash in ice-cold PBS, cells were lysed. Lysates were separated by SDS- diluted in culture medium just before use. PAGE electrophoresis and immunoblotted using anti–phospho- In vitro assays using transduced Ba/F3 cells. The retroviral expres- PDGFRA(Tyr754), anti-PDGFRA, and anti-actin antibodies. D sion constructs pMSCV-PDGFRAD842V and pMSCV-PDGFRA DIM842-844 The 5-bromo-2¶-deoxyuridine cell proliferation (Roche Applied were described previously (12). Retrovirally transduced Ba/F3 cells, Science) and sulforhodamine B survival (Sigma-Aldrich) assays were stably expressing the above PDGFRA constructs, were maintained in used to evaluate the proliferation rate and the survival of primary GIST culture at 1 Â 106 cells/mL. For the Ba/F3 dose-response assays, cells, according to the manufacturer’s instructions (18, 19). Both tests retrovirally transduced Ba/F3 cells were grown in RPMI 1640 supple- were run simultaneously and in duplicate. In short, the adherent mented with 10% fetal bovine serum, with or without interleukin 3, primary GIST cells were seeded in 96-well plates (10,000 cells per well) in the presence of vehicle alone or with varying concentrations of the and incubated overnight at +37jC.