ResearchMotesanib inhibits Kit mutations associated with gastrointestinal stromal tumors

Background: Activating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST). Methods: This study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Singleand double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays. Results: Motesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Δ552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays. Conclusions: In conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations. Background Approximately 85% to 90% of all cases of gastrointestinal stromal tumors (GIST) are associated with gain-of-function mutations in the gene KIT [1-4]. A further 5% to 10% of cases of GIST are associated with activating mutations in the platelet-derived growth factor receptor alpha (PDGFRα) gene [1,4,5]. Activating Kit mutations in GIST occur principally in the extracellular domain, the juxtamembrane domain (which regulates receptor dimerization), kinase domain I, and kinase domain II (or activation loop) [1]. Imatinib, a small-molecule inhibitor of Kit and PDGFRα, represents an effective first-line therapy option for patients with advanced GIST [6]. Imatinib is a potent inhibitor of wild-type Kit and juxtamembrane domain Kit mutants, while Kit activation loop mutants are resistant [1,7]. Secondary imatinib resistance is most commonly associated with the acquisition of a secondary mutation in Kit (either in the kinase domain I or the activation loop) or in PDGFRα [8]. Motesanib is an orally administered small-molecule antagonist of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR and Kit [9,10]. In clinical studies, motesanib has shown encouraging efficacy in the treatment of patients with advanced solid tumors [1013]. In biochemical assays, motesanib potently inhibits the activity of both Kit (50% inhibitory concentration [IC50] = 8 nM) and PDGFR (IC50 = 84 nM) [9], suggesting that it may have direct antitumor activity in GIST [14,15]. The aim of this study was to characterize the ability of motesanib to inhibit the activity of wild-type Kit in vitro and in vivo, and to investigate differences in the potency of motesanib and imatinib against clinically important primary activating Kit mutants and mutants associated with secondary imatinib resistance. The results suggest that motesanib has inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations. * Correspondence: phughes@amgen.com 1 Department of Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA, 91320-1799, USA Full list of author information is available at the end of the article © 2010 Caenepeel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Caenepeel et al. Journal of Experimental & Clinical Cancer Research 2010, 29:96 http://www.jeccr.com/content/29/1/96 Page 2 of 8 Methods Reagents Unless specified otherwise all reagents were purchased from Sigma Aldrich; all cell culture reagents were purchased from Invitrogen (Carlsbad, CA). In Vivo Hair Depigmentation Assay Female C57B6 mice (6 to 8 weeks old; 20 to 30 g; Charles River Laboratories, Wilmington, MA) were anesthetized, and an area of skin 2 × 2 cm on the right flank was depilated. Oral administration of either 75 mg/kg motesanib (Amgen Inc., Thousand Oaks, CA) or vehicle (water, pH 2.5) was initiated on the same day as depilation and continued for 21 days. On day 21, photographs were taken for assessment of hair depigmentation. The same patch of skin was depilated again on day 28, and photographs for assessment of depigmentation were taken on day 35. All animal experimental procedures were conducted in accordance with the guidelines of the Amgen Animal Care and Use Committee and the Association for Assessment and Accreditation of Laboratory Animal Care standards. Preparation of Wild-Type and Mutant KIT Constructs KIT mutants (Table 1) were identified from published reports [8] and generated using PCR-based site-directed mutagenesis. PCR products were cloned into the pcDNA3.1+ hygro vector or the pDSRα22 vector (Amgen Inc), gel purified, and then ligated with a common 5' fragment of human wild-type KIT to yield full-length, mutant constructs in pcDNA3.1+ hygro or pDSRα22 expression vectors. Stable Transfection of CHO and Ba/F3 Cells With Wild-Type and Mutant KIT AM-1/D Chinese Hamster Ovary (CHO) cells (Amgen Inc.) were maintained under standard conditions. Cells were transfected with wild-type or mutant KIT using Lipofectamine2000 and Opti-MEM (Invitrogen) following the manufacturer's instructions. Four days after transfection, cells were transferred into selection medium: Gibco DMEM High Glucose with 10% FBS plus 300 μg/ mL hygromycin (Roche Applied Sciences, Indianapolis, IN) for cells transfected with pcDNA3.1+ hygro; DMEM High Glucose with 10% dialyzed FBS for cells transfected with pDSRα22. Stably transfected CHO cells were selected 2 weeks later and maintained as described above. Interleukin 3 (IL-3)-dependent Ba/F3 cells were maintained under standard conditions including 3 ng/mL murine IL-3 (Cat # PMC0035; Invitrogen/BioSource). Cells were transfected with wild-type or mutant KIT in the pDSRa22 expression vector along with linearized pcDNA Neo using the Nucleofector Kit V and a Nucleoporator (Lonza; Cologne, Germany) following the manufacturer's instructions. Two to 3 days post transfection, cells were transferred into selection medium (supplemented RPMI medium plus 750 μg/mL G418). Stably transfected Ba/F3 cells were maintained in supplemented RPMI medium plus 3 ng/mL murine IL-3. Fluorescence activated cell sorting (FACS) was utilized to isolate pools of CHO and Ba/F3 cells stably expressing wild-type and mutant KIT variants. FACS was performed on a FACS Aria cell sorter (BD Biosciences San Jose, CA), under sterile conditions using 488 nm laser excitation. KIT transfected cells were labeled with the anti-Kit monoclonal antibody SR1 (prepared at Amgen Inc.; data on file) followed by incubation with FITC-labeled secondary anti-mouse IgG antibody (SouthernBiotech, Birmingham, AL). Cells were then resuspended in Dulbecco's phosphate-buffered saline with 0.5% bovine serum albumin at a final concentration of 1 × 106 cells per mL to ensure a constant and viable sorting rate of 5000 cells/sec. Cells transfected with vector control were used to adjust the baseline instrument settings. Forward and side scatter gating enabled the exclusion of dead cells and debris. The top 10% to 15% of Kit-positive cells within the overall transfected cell population were then isolated to ensure collection of high-expressing cells. Cells were Table 1: Clinically Relevant KIT Mutations KIT Genotype Mutation Type Domain Primary activating mutations Δ552-559 Deletion Juxtamembrane domain V560D Single mutation Juxtamembrane domain AYins503-504 Insertion Extracellular domain Secondary imatinib-refractory mutations D816V Single mutation Activation loop Y823D Single mutation Activation loop V560D/V654A Double mutation Juxtamembrane domain/kinase domain I V560D/T670I Double mutation Juxtamembrane domain/kinase domain I Caenepeel et al. Journal of Experimental & Clinical Cancer Research 2010, 29:96 http://www.jeccr.com/content/29/1/96 Page 3 of 8 sorted directly into 15 mL conical tubes containing the appropriate growth media. Cell pools were then cultured and maintained under the respective selection conditions, and were reanalyzed for Kit expression prior to characterization of Kit autophosphorylation. Cell-Based Kit Autophosphorylation Assay CHO cells stably transfected with wild-type or mutant isoforms of KIT were seeded in a 96-well tissue culture plate at a density of 2 × 104 cells per well. For stem cell factor (SCF) characterization experiments, cells were stimulated with serial dilutions of SCF for varying times. To determine IC50 values, the cells were treated for 2 hours with single 10-fold serial dilutions of motesanib or imatinib starting at 3 μM. Cell lines transfected with wild-type KIT were stimulated for 10 minutes with 100 ng/mL SCF following treatment with motesanib or imatinib. Cell lines transfected with activating KIT mutants were not stimulated with SCF in IC50 experiments. Cells were washed with phosphate-buffered saline and lysed in RIPA buffer (50 mM Tris, pH 7; 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 300 μM activated sodium vanadate, 1× protease inhibitor cocktail) for 30 minutes at 4°C with shaking. Cell lysates were added to a 96-well DELFIA microplate (PerkinElmer Inc.) coated with anti-Kit antibody (1 μg per well; AF332, R&D Systems, Inc.; Minneapolis, MN) and incubated for 2 hours. Lysates were then removed and the plate was washed 3 times with DELFIA wash buffer (PerkinElmer Inc.). Recombinant anti-phosphotyr