Resistance to C-KIT Kinase Inhibitors Conferred by V654A Mutation

1159

Resistance to c-KIT kinase inhibitors conferred by V654A mutation

Kathryn G. Roberts,1 Adam F. Odell,1 V654A c-KIT. The findings were supported by studies of Ellen M. Byrnes,1 Rosa M. Baleato,1 c-KIT phosphorylation. Analysis of the crystal structure Renate Griffith,2 Alan Bruce Lyons,3 of imatinib in complex with the kinase domain of c-KIT and Leonie K. Ashman1 predicts that the V654A substitution directly affects the binding of imatinib to the receptor. Alternative c-KIT 1School of Biomedical Sciences and 2School of Environmental inhibitors, nilotinib (AMN107) and PKC412, were also and Life Sciences, University of Newcastle, and Hunter Medical less active on V560G/V654A c-KIT than on the V560G Research Institute, Newcastle, New South Wales, Australia; single mutant; however, nilotinib, like imatinib, potently and 3Division of Haematology, Hanson Institute, Institute of Medical and Veterinary Sciences, Adelaide, South Australia, inhibited the V560G mutant. PKC412 strongly inhibited Australia imatinib-resistant D816V c-KIT. [Mol Cancer Ther 2007;6(3):1159–66] Abstract Certain mutations within c-KIT cause constitutive activa- Introduction tion of the receptor and have been associated with several c-KIT is classified as a type 3 receptor tyrosine kinase and is human malignancies. These include gastrointestinal stro- structurally similar to platelet-derived growth factor mal tumors (GIST), mastocytosis, acute myelogenous receptor, c-FMS, and FLT-3 (1). Distinguishing features of leukemia, and germ cell tumors. The kinase inhibitor this family include an extracellular region composed of imatinib potently inhibits c-KIT and is approved for five immunoglobulin-like loops, which is followed by a treatment of GIST. However, secondary point mutations single hydrophobic transmembrane domain that anchors can develop within the kinase domain to confer resistance the receptor to the plasma membrane. The intracellular to imatinib and cause drug-resistant relapse. A common region contains a negative-regulatory juxtamembrane mutation, which results in a V654A substitution, has been domain (JMD) and a tyrosine kinase split by an interkinase documented in imatinib-resistant GIST patients. We sequence into two regions, the ATP binding pocket and the c-KIT expressed cDNA constructs encoding the V654A phosphotransferase catalytic site. Binding of the cognate substitution alone and in combination with a typical ligand, stem cell factor (SCF), induces receptor dimeriza- activating exon 11 mutation characteristic of GIST, tion and autophosphorylation of specific tyrosine residues V560G, in factor-dependent FDC-P1 cells. The V654A within the cytoplasmic domain. This creates docking sites substitution alone resulted in enhanced proliferation in for signal transduction molecules, which activate down- c-KIT ligand (stem cell factor) but not factor indepen- stream pathways that regulate proliferation, survival, dence. Cells expressing the double mutant were, like those adhesion, migration, and differentiation (1). c-KIT is expressing single V560G mutant c-KIT, factor indepen- expressed in hematopoietic precursors, mast cells, germ dent. Analysis of cellular proliferation in the presence of cells, melanocytes, and interstitial cells of Cajal that reside imatinib showed that the V654A substitution alone within the gastrointestinal tract (2). conferred resistance. The difference in sensitivity was Specific mutations within the c-KIT gene cause constitu- especially pronounced for cells expressing single mutant tive phosphorylation and activation of the kinase domain V560G c-KIT compared with double mutant V560G/ resulting in uncontrolled cell proliferation. Such genetic abnormalities have been implicated in the pathogenesis of gastrointestinal stromal tumors (GIST), mastocytosis, as well as some cases of acute myelogenous leukemia and Received 10/17/06; revised 12/13/06; accepted 1/24/07. testicular seminoma (3). The first ‘‘gain-of-function’’ muta- Grant support: National Health and Medical Research Council of Australia grant 150413 (L.K. Ashman) and the Anthony Rothe Memorial Trust. tions in c-KIT were discovered in the human mast cell New South Wales Health via the Hunter Medical Research Institute leukemia cell line HMC-1 (4). These cells contain a wild- (infrastructure support). type (WT) c-KIT allele and an allele that harbors two point The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked mutations, which result in amino acid substitutions V560G advertisement in accordance with 18 U.S.C. Section 1734 solely to in the intracellular JMD, and D816V within the activation indicate this fact. loop of the kinase domain. Expression of the corresponding Note: K.G. Roberts and A.F. Odell contributed equally to this work. mouse c-KIT mutants, V559G and D814V, in mouse factor- Requests for reprints: Leonie K. Ashman, University of Newcastle, Room dependent hematopoietic cell lines or normal bone marrow 511, Medical Sciences Building, Callaghan, New South Wales, 2308 Australia. Phone: 61-2-4921-7947; Fax: 61-2-4921-6903. cells resulted in constitutive tyrosine phosphorylation, E-mail: [email protected] kinase activity, factor-independent growth, and tumorige- Copyright C 2007 American Association for Cancer Research. nicity in mice (5–7). The D816V substitution has been doi:10.1158/1535-7163.MCT-06-0641 identified in peripheral blood mononuclear cells of patients

Mol Cancer Ther 2007;6(3). March 2007

suffering from mastocytosis with an associated hematologic We have examined the molecular mechanisms underly- disorder (8). Corresponding mutations at codon 816 ing the resistance to imatinib by expressing c-KIT contain- have also been detected in acute myelogenous leukemia ing the V564A substitution alone and in combination with (9, 10) and germ cell tumors (11). c-KIT mutations in acute a typical activating mutation found in GIST, V560G, in myelogenous leukemia are largely confined to ‘‘core mouse factor-dependent FDC-P1 cells. Sensitivity of these binding factor’’ leukemias that are characterized by t(8;21) mutant forms to imatinib and newly developed c-KIT kinase or inv(16) (refs. 12, 13). Mutations have been shown to inhibitors nilotinib (AMN107) and PKC412 was examined occur at multiple locations in the protein, with mutations at in vitro and compared with the well-characterized imatinib- codon 816 accounting for about half (13, 14). Mutations resistant D816V c-KIT mutant. within c-KIT are detected on f80% of GIST specimens, which arise from transformation of the interstitial cells of Materials and Methods Cajal. Most mutations are located within the intracellular c-KIT Expression in FDC-P1Cells JMD and include in-frame deletions and insertions of The nonadherent mouse factor-dependent early myeloid various sizes along with point mutations that result in cell line, FDC-P1, was maintained in DMEM containing 10% amino acid substitutions (15). In addition, a small propor- FCS and 25 units/mL mouse granulocyte macrophage-CSF tion of c-KIT mutations have also been detected within the (GM-CSF). FDC-P1 lines expressing WT, V560G, and D816V extracellular JMD and kinase regions (16). mutant forms of human c-KIT have been described The phenylaminopyrimidine compound, imatinib, selec- previously (34). To create the V654A mutation, QuikChange tively regulates the activity of certain tyrosine kinases, Site-Directed Mutagenesis (Stratagene, La Jolla, CA) was in particular BCR-ABL, platelet-derived growth factor done on WT c-KIT in pBluescript. The oligonucleotide receptor, and c-KIT, through competitive inhibition at the primers (Sigma Genosys, Castle Hill, New South Wales, ATP binding site. Although it binds with lower affinity, Australia) were as follows: 5¶-CTTGGTAATCACATGAA- imatinib is also active against the colony-stimulating factor TATTGCCAATCTACTTGGAGCC-3¶ (forward) and 5¶- (CSF)-1 receptor, c-FMS, at therapeutically attainable GGCTCCAAGTAGTTGGCAATATTCATGTGATTAC- concentrations (17). Imatinib interacts with the inactive CAAG-3¶ (reverse). A 159-bp fragment spanning the V654A conformation of the kinase domain and locks it into this mutation was excised with PacI and ApaI and directionally position (18, 19). As a consequence, the transfer of phos- cloned into the corresponding restriction sites in WT c-KIT phate groups to substrate molecules is prevented, and in the defective retroviral expression vector pRUFneo. The downstream signaling cascades normally generated by V560G/V654A double mutant was generated by excising the kinase activation are inhibited (20). Clinical trials for the PacI and ApaI fragment from pBluescript-V654A c-KIT and treatment of chronic myelogenous leukemia and BCR- ligating it into the corresponding restriction sites of ABL–positive acute lymphoid leukemia showed superior pRUFneo-V560G c-KIT. efficacy and safety compared with traditional chemother- Both constructs were verified by automated DNA apeutic agents (21). In vitro studies showed that imatinib sequencing and transfected into BOSC 23 packaging cells also inhibits the kinase activity of c-KIT (22). Promising (35) using LipofectAMINE 2000 (Invitrogen, Mount results reported from phase I and II clinical trials led to Waverly, Victoria, Australia). Forty-eight hours posttrans- the approval of imatinib for the treatment of malignant fection, the retroviral supernatants were harvested and metastatic and/or unresectable GIST (23). filtered through a 0.45 Am filter. Retroviral infection was Despite the clinical success of imatinib in the treatment of done by resuspending 106 FDC-P1 cells in a suspension GIST, numerous reports are confirming the emergence of containing 3 mL DMEM/10% FCS and 1 mL retroviral resistance in secondary progressive tumors. This phenom- supernatant with 8 Ag/mL polybrene (Chemicon, Teme- enon of acquired resistance in GIST patients recapitulates cula, CA). After 48 h, the infectants were selected with treatment failure observed in advanced chronic myeloge- 1 mg/mL G418 until uninfected control cells were dead. nous leukemia patients (24, 25) and is a major setback that FDC-P1 (V654A KIT) were further selected by growth in must be addressed when developing targeted protein 100 ng/mL recombinant human SCF (Peprotech, Rocky kinase inhibitors. As with BCR-ABL in chronic myeloge- Hill, NJ). G418-resistant FDC-P1 (V560G/V654A KIT) cells nous leukemia, the tumors from the majority of these were further selected for growth in the absence of relapsing patients displayed new secondary point muta- GM-CSF. In both cases, the introduced c-KIT sequences tions in the exons of the c-KIT gene corresponding to the were confirmed to be correct by isolation of genomic DNA kinase domain (26–30). These missense mutations are from the cells following selection, PCR of the retroviral believed to alter the structure of the kinase domain and insert, and sequencing. FDC-P1 (WT KIT) and FDC-P1 consequently interfere with interactions between imatinib (V654A KIT) were routinely maintained in mouse GM-CSF and the receptor (31). In particular, the occurrence of a (as above). FDC-P1 (D816V KIT), FDC-P1 (V560G KIT), and missense mutation that results in the substitution of valine FDC-P1 (V560G/V654A KIT) were maintained in the by alanine at codon 654 (V654A) has been documented in absence of factor. All cell populations were assessed for rapidly progressive imatinib-resistant samples from GIST c-KIT expression by flow cytometry and Western blotting. À patients in several studies (26–30, 32) and is the most All c-KIT constructs encode the more abundant GNNK common mutation associated with resistance (33). isoform (34).

MolCancerTher2007;6(3).March2007

c-KIT Kinase Inhibitory Drugs tyrosine monoclonal antibodies 4G10 (Upstate Biotechnolo- Imatinib was repurified from capsules obtained commer- gy, Lake Placid, NY) and PY20 (BD Biosciences) followed by cially (Novartis, Basel, Switzerland). Nilotinib (AMN107) horseradish peroxidase–labeled secondary antibody. Mem- and PKC412 were provided by Novartis. branes were stripped in stripping buffer [62.5 mmol/L Tris Immunofluorescence Assay (pH 6.5), 100 mmol/L 2-mercaptoethanol, 0.1% SDS] at 60jC Cell surface expression of c-KIT on FDC-P1 cells was for 30 min with agitation and reprobed for c-KIT using goat examined by indirect immunofluorescence as described polyclonal anti–c-KIT antibodies (Santa Cruz Biotechno- previously (36) and analyzed using a FACSCalibur flow logy, Santa Cruz, CA). Immunoblots were visualized with cytometer (BD Biosciences, San Jose, CA). enhanced chemiluminescence plus reagent on Typhoon Cell Proliferation Assay Imaging System (Amersham Biosciences) and quantified To evaluate cell survival and growth, the 3-(4-5-dime- with ImageQuant software. thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo- Structural Analysis phenyl)-2H-tetrazolium salt (MTS) assay was used. FDC- The X-ray crystallographic structures of c-KIT in the P1 cell lines were cultured in 96-well trays at a density of active and inactive conformations and complexed with 2 Â 105/mL in DMEM/10% FCS containing appropriate imatinib (37, 38) were downloaded from the internet-based factors. In some experiments, imatinib, nilotinib, or Research Collaboratory for Structural Bioinformatics Pro- PKC412 was added to cultures at the indicated concen- tein Data Bank4 (accession nos. 1T45, 1T46, and 1PKG). trations. After 48 h, viable cells were measured using the The structures were analyzed in DS Viewer Pro (Accelrys, CellTiter96 Aqueous Cell Proliferation Assay (Promega, Inc. San Diego).5 Madison, WI). Immunoprecipitation and Western Blot Analysis Results FDC-P1 cells expressing WT or mutant forms of c-KIT Expression Levels ofc-KIT in FDC-P1 Cells grown in log phase were serum and, where appropriate, FDC-P1 cells were infected with retrovirus encoding growth factor starved in DMEM supplemented with 0.1% human V654A c-KIT or V654A/V560G c-KIT and then Â 6 j bovine serum albumin at 2 10 /mL for 2 h at 37 C. Cells incubated in G418 to select for successfully transduced were incubated in the presence or absence of various cells. V654A c-KIT–expressing cells were further selected concentrations of imatinib, nilotinib, or PKC412 as indicated. for growth in human SCF in place of mouse GM-CSF. The Â 7 Cells were resuspended to 2 10 /mL in serum-free DMEM V654A/V560G c-KIT–expressing cells were selected for and, where indicated, stimulated with 100 ng/mL SCF factor-independent growth. The other cell lines were as at 37jC for 5 min followed by the addition of ice- Â described previously (34). The level of c-KIT on the surface cold PBS. Cells were pelleted at 220 g and lysed in 1.2 mL of FDC-P1–derived cell lines was investigated using of modified radioimmunoprecipitation assay buffer [1% immunofluorescence and flow cytometric analysis (Fig. 1). NP40, 50 mmol/L Tris (pH 7.6), 150 mmol/L NaCl, The relative fluorescence of each cell line was compared 1 mmol/L EDTA, 0.25% sodium deoxycholate, 0.05% SDS] with a negative antibody control. The results indicated that supplemented with 5 mmol/L sodium fluoride, c-KIT was expressed at relatively high levels on FDC-P1 5 mmol/L tetrasodium pyrophosphate, 5 mmol/L sodium (WT KIT) and FDC-P1 (V560G KIT) cells. FDC-P1 (V654A vanadate, 1 mg/mL leupeptin, 1 mg/mL aprotinin, 1 mmol/L KIT) and FDC-P1 (V560G/V654A KIT) cells displayed phenylmethylsulfonyl fluoride, and Complete Protease moderate levels, which contrasted with the relatively low Inhibitor Cocktail (Roche, Mannheim, Germany) and incu- surface expression of the D816V mutant c-KIT. bated on ice for 30 min. Lysates were clarified by Â Preliminary Studies withV654A Mutant c-KIT centrifugation at 15,000 g for 15 min, and total protein To confirm that the V654A substitution was capable of content was determined by bicinchoninic acid analysis conferring imatinib resistance in this cell line model, FDC- (Pierce Biotechnology, Rockford, IL). For immunoprecipita- P1 cells expressing WT or V654A c-KIT and maintained in tion, 1 mg total protein from each lysate, or 3 mg total protein SCF were tested for growth in the presence of increasing for immunoprecipitation of D816V c-KIT, was incubated A concentrations of imatinib by MTS assay, which quantitates with 4 g antihuman c-KIT monoclonal antibody KIT4 and the number of viable cells after a 48-h incubation period. 25 Al of a 50% slurry of protein A-Sepharose (Amersham j The experiment also included imatinib-resistant FDC-P1 Biosciences, Castle Hill, New South Wales, Australia) at 4 C (D816V KIT) cells for comparison. The results, shown in on a rotating platform for 2 h. Immunoprecipitates were Fig. 2A, confirmed that, under these conditions, the V654A washed four times in lysis buffer supplemented with 5 mutation conferred a high degree of resistance to the mmol/L sodium orthovanadate and 5 mmol/L sodium drug. The proliferation of FDC-P1 (WT KIT) cells was fluoride. After the final wash, the protein A-Sepharose pellet inhibited by imatinib with a IC of 0.1 to 0.2 Amol/L as was resuspended in loading buffer containing 2-mercaptoe- 50 reported previously (34). In contrast, FDC-P1 (V654A KIT) thanol, boiled for 5 min, and subjected to SDS-PAGE on 8% gels. Gels were transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham Bio- sciences), blocked with 1% bovine serum albumin in TBS 4 http://www.rcsb.org containing 0.1% Tween 20, and probed with anti-phospho- 5 http://www.accelrys.com

Mol Cancer Ther 2007;6(3). March 2007

Effect of the V654A Substitution in Combination with V560G, an Activating Mutation Characteristic of GIST,onDrugSensitivity Clinically, the V654A substitution occurs in the context of GIST with primary activating mutations in exon 9 or exon 11, resulting in relapse of the disease in patients treated with imatinib. Exon 11 encodes the negative-regulatory intracellular JMD of c-KIT, and such mutations greatly increase sensitivity to imatinib due to increased access to its binding site (34, 38, 40, 41). In comparison with the V560G single mutant, FDC-P1 cells expressing the V560G/V645A mutant were much more resistant (approximate imatinib IC50 of 150 nmol/L compared with 15 nmol/L) and more resistant than the cells expressing WT c-KIT (Fig. 3B). The

Figure 1. Cell surface expression of c-KIT on FDC-P1 cells. Surface expression levels of c-KIT were determined by indirect immunofluorescence and flow cytometry using anti-human c-KIT monoclonal antibody (1DC3; bold line). An isotype-matched negative control monoclonal antibody (IB5) was used to determine background immunofluorescence (thin line).

and FDC-P1 (D816V KIT) were resistant to inhibition by A imatinib and in both cases displayed a IC50 of >5 mol/L. This growth rate was significantly increased compared with FDC-P1 (WT KIT; P < 0.05, Student’s t test, three independent experiments). In addition, FDC-P1 (V654A KIT) cells were able to form colonies in the presence of 1 Amol/L imatinib in the semisolid medium methylcellu- lose, whereas cells expressing WT c-KIT did not (data not shown). Certain oncogenic mutant forms of c-KIT, such as the D816V mutant, are intrinsically resistant to imatinib because they strongly favor the active conformation of the kinase domain, which cannot bind the drug (39). These activating mutations induce factor-independent growth of FDC-P1 cells. To assess this characteristic, FDC-P1–derived cell lines, as above, were cultured in increasing concentrations of SCF for 48 h and proliferation was evaluated by MTS assay. As expected, FDC-P1 (WT KIT) cells did not survive in the absence of factor, and their proliferation was dependent on increasing concentrations of SCF (Fig. 2B). In contrast, the proliferation of FDC-P1 (D816V KIT) cells remained constant, regardless of SCF concentration. Inter- Figure 2. Properties of FDC-P1 cells expressing V654Amutant c-KIT. estingly, the growth of FDC-P1 (V654A KIT) cells at low A, proliferation in the presence of imatinib. FDC-P1 expressing WT, concentrations of SCF ranging from 1.5 to 12 ng/mL V654A, and D816V c-KIT were incubated for 48 h in the presence of imatinib at the indicated concentrations. Cultures of FDC-P1 (WT KIT) and differed significantly from FDC-P1 cells expressing WT FDC-P1 (V654AKIT) contained 100 ng/mL SCF. Cell proliferation was c-KIT (P < 0.05, Student’s t test, three independent measured by MTS assay. B, SCF dependence. FDC-P1 cells as above were experiments). However, the proliferation of these cells cultured for 48 h in the indicated concentrations of SCF and proliferation Points, was still dependent on SCF. This indicated that the V654A was determined by MTS assay. mean of three individual experiments, where each determination was done in quadruplicate; bars, SE. *, mutation caused increased proliferation at low concen- P < 0.05, statistically significant differences between FDC-P1 expressing trations of SCF but did not confer constitutive activity. WT and V654Ac-KIT, t test, n =3.

MolCancerTher2007;6(3).March2007

Figure 3. Effect of imatinib, nilotinib, and PKC412 on proliferation of FDC-P1 cells expressing WT, V654A, V560G, V560G/V654A, and D816V mutant c-KIT. Cells were incubated for 48 h in inhibitors at the indicated concentrations, and cell number was determined by MTS assay. Cultures of FDC-P1 (WT KIT) and FDC-P1 (V654AKIT) contained 100 ng/mL SCF; all other cultures were factor independent. Results are expressed as percentage of control with no added inhibitor. Points, mean of six replicates; bars, SE.

greater sensitivity of cells expressing the V654A mutant resistance to nilotinib, although slight inhibition was seen alone in these experiments compared with those shown in at the highest level tested (500 nmol/L). Nilotinib did not Fig. 2 was found to be due to taking cells directly from affect the growth of cells in GM-CSF except for cells maintenance in GM-CSF (i.e., without preconditioning in expressingthedoublemutantwhereinhibitionwas SCF) into the inhibitor assays. This has no effect on the reproducibly seen at the highest level of drug (Fig. 3C). results obtained with cells expressing the constitutively In contrast, the staurosporine derivative PKC412 was much active mutants, which were grown in the absence of added less specific, displaying considerable inhibition of cell growth factors throughout. In all cases, cells cultured in survival and/or proliferation of all cell lines irrespective GM-CSF, and consequently independent of c-KIT signaling, of whether growth was dependent on GM-CSF or c-KIT were minimally affected by imatinib (Fig. 3A). signaling (Fig. 3E). For c-KIT–dependent growth, in The inhibitory effects of alternative inhibitors of c-KIT, contrast to imatinib and nilotinib, the activating V560G nilotinib and PKC412, were tested to determine whether mutation had a minimal effect on sensitivity. Similarly, the the V654A mutation also caused resistance to these drugs. V654A mutation did not appreciably affect sensitivity to As shown in Fig. 3D, V560G mutant c-KIT was more PKC412. The notable feature of the PKC412 data was the sensitive to nilotinib than was WT c-KIT, a result similar to sensitivity of the kinase domain mutant, D816V, to this that with imatinib. Nilotinib sensitivity of cells expressing drug (Fig. 3F). the V560G/V564A mutant was reduced compared with Effect of Kinase Inhibitors on c-KIT Phosphorylation those harboring the V560G mutant, although they c-KIT–expressing FDC-P1 cells were incubated in the remained more sensitive than cells expressing WT c-KIT. presence of various concentrations of imatinib, nilotinib, or The effect of the V654A mutation alone was less pro- PKC412 for 2 h under serum-free conditions and then lysed nounced than for imatinib. As with imatinib, the D816V and immunoprecipitated with c-KIT antibody. FDC-P1 mutant kinase domain mutation conferred a high level of (WT KIT) and FDC-P1 (V654A KIT) cells were pulsed with

Mol Cancer Ther 2007;6(3). March 2007

SCF for 5 min before lysis. Immunoprecipitates were alone. In contrast to the proliferation results, phosphoryla- electrophoresed, then Western blotted, and analyzed for tion of V560G c-KIT was less inhibited by PKC412 than tyrosine-phosphorylated and total c-KIT (Fig. 4A). Data WT c-KIT, particularly in the double mutant. As reported were quantitated and phosphorylated receptor levels previously, phosphorylation of D816V mutant c-KIT was were adjusted for total c-KIT and expressed as a percentage not influenced by imatinib up to 1 Amol/L. It was weakly of control phosphorylation in the absence of inhibitor inhibited by nilotinib and potently inhibited by PKC412. (Fig. 4B). The results generally supported the cell proliferation data with the V654A substitution decreasing imatinib sensitivity Discussion in the context of both WT and V560G mutant c-KIT. Like It has become apparent that point mutations in the kinase imatinib, nilotinib (but not PKC412) more potently domain are a major mechanism of drug-resistant relapse in inhibited V560G c-KIT than WT c-KIT phosphorylation. cancer patients treated with small-molecule protein kinase However, inhibition by nilotinib was less influenced by the inhibitors (31, 42). For inhibitors, such as imatinib that V654A substitution, especially in the context of WT c-KIT. target the inactive conformation of the kinase (19, 38), PKC412 inhibition was not influenced by the V654A change two general mechanisms can lead to resistance (43). First,

Figure 4. Phosphorylation of c-KIT in the presence of inhibitors. A, FDC-P1 cells expressing WT or mutant c-KIT as for Fig. 3 were serum starved for 2 h in the presence of the indicated concentrations of imatinib (Ima), nilotinib (Nil), or PKC412 (PKC). FDC-P1 (WT KIT) and FDC-P1 (V654AKIT) were stimulated with 100 ng/mL SCF for the final 5 min. Cells were lysed in radioimmunoprecipitation assay buffer and cleared lysates were immunoprecipitated with c-KIT antibody and analyzed by SDS-PAGE and Western blotting (WB) onto nitrocellulose membrane. Blots were probed with anti-phosphotyrosine (PY20 and 4G10), then stripped, and reprobed with anti – c-KIT antibodies. Upward smearing of the bands is due to c-KIT ubiquitylation following activation (e.g., ref. 36). B, phosphotyrosine signals were quantified using ImageQuant software, corrected for total c-KIT, and expressed as a percentage of the values obtained in the absence of inhibitor. Columns, mean of three independent experiments; bars, SD.

MolCancerTher2007;6(3).March2007

mutations that strongly favor the active conformation of the pyrimidine compound related to imatinib. It has shown kinase domain can preclude drug binding by an indirect efficacy in imatinib-resistant chronic myelogenous leuke- (conformational) mechanism. An example of this is the mia associated with several kinase domain mutants of D816V substitution in the activation loop of the kinase BCR-ABL (46). Like imatinib, nilotinib potently inhibited domain of c-KIT (39). Such mutant receptors confer consti- JMD mutant (V560G) c-KIT in both autophosphorylation tutive kinase activity and the ability to grow in the absence and cell proliferation assays. In contrast to imatinib, it of added growth factors on normally factor-dependent inhibited V560G/V654A mutant c-KIT to a similar extent to cells (34, 43). Second, kinase domain mutations that directly WT c-KIT in both assays. These two features suggest that affect drug binding can confer resistance. Examples of nilotinib would be highly suitable for treatment of GIST this are the T315I substitution in ABL and the homologous with exon 11 mutant c-KIT and may be less susceptible to T670I substitution in c-KIT (24, 29). Such mutations do not resistance arising from the V654A mutation. Other studies necessarily result in constitutive kinase activity. have reported varying results on the efficacy of nilotinib The V654A c-KIT kinase domain mutation has been in inhibiting activation loop mutant D816V c-KIT (47, 48). associated with imatinib-resistant GIST relapse in several In our hands, this mutant displayed increased sensitivity to studies (26–30, 33). We have examined this substitution in nilotinib compared with imatinib, but this was insufficient the context of WT c-KIT to better characterize its specific to be of likely therapeutic value. The indolinine inhibitor functional effects. We confirmed V654A c-KIT resistance to sunitinib (SU11248) was also shown to inhibit c-KIT with imatinib using cell proliferation and c-KIT autophosphor- exon 11 + V654A mutations in GIST cells treated ex vivo and ylation assays. The V654A mutation did not lead to consti- in Ba/F3 cells (49). In the GIST specimen, the inhibition was tutive kinase activity in either the receptor phosphorylation less than observed in another specimen with a distinct exon assay (data not shown) or the cell proliferation assay, 11 mutation alone, suggesting that the activity of this drug although it did enhance the response to low levels of SCF may also be reduced by the secondary V654A mutation. (Fig. 2B). This suggests that the V654A substitution did not However, direct comparisons with c-KIT having the same markedly favor the active conformation of the kinase primary mutation and also with WT c-KIT in the same domain but rather conferred resistance primarily by cellular background are required. directly impeding imatinib binding. The V654 side chain The staurosporine derivative, PKC412, displayed more contacts imatinib directly in the crystal structure (38), and cytotoxicity unrelated to c-KIT inhibition in the cell the loss of this hydrophobic interaction in the V654A proliferation assay and was a weaker inhibitor of V560G mutant is predicted to decrease imatinib binding affinity. and V560G/V654A c-KIT than the other two compounds in As published recently by others (32, 44), our modeling both assays. In contrast, others found that the drug did confirmed this prediction (data not shown). In addition, the inhibit phosphorylation of c-KIT Y703 in GIST cells imatinib resistance of V654A c-KIT may be due in part to an harboring the V654A mutation together with an exon 9 enhanced ability to adapt the active conformation. This mutation (27). The differences may be due to the different is manifested in the increased sensitivity of the mutant to conformational effects of the exon 9 and exon 11 mutations low concentrations of SCF and can also be rationalized by on PKC412 binding. Notably, PKC412 was an effective analysis of the c-KIT crystal structures (37, 38). Amino acid inhibitor of D816V mutant c-KIT. Inhibition of D816 mutant 654 is located on a loop directly before the aC helix. This c-KIT by PKC412 has also been reported by others (40, 48, helix must move considerably to attain its position in the 50), and the drug holds promise for the treatment of cases active conformation. We suggest that the less bulky alanine of mastocytosis and acute myelogenous leukemia harbor- side chain allows greater freedom of movement in this part ing this mutation. of the protein, thus facilitating conformational change. Overall, the data further show the need for careful mapping The majority of GIST have mutations in exon 11 affecting of mutations in target kinases for selection of the most the negative-regulatory intracellular JMD of c-KIT, whereas appropriate drugs for individual patients. smaller proportions have mutations in exon 9 encoding the extracellular juxtamembrane region or in platelet-derived growth factor receptor a (16). Exon 11 mutations confer References enhanced sensitivity to imatinib (34, 41), and patients with 1. Lennartsson J, Jelacic T, Linnekin D, Shivakrupa R. Normal and oncogenic forms of the receptor tyrosine kinase kit. Stem Cells 2005;23: these mutations have the best response to imatinib therapy 16 – 43. (45). Because the exon 13 V654A mutation seems to confer 2. Ashman LK. The biology of stem cell factor and its receptor C-kit. Int J imatinib resistance in GIST, we have also examined this Biochem Cell Biol 1999;31:1037 – 51. substitution in combination with the JMD V560G (exon 11) 3. Kitamura Y, Hirota S. Kit as a human oncogenic tyrosine kinase. Cell mutation. The double mutant displayed reduced imatinib Mol Life Sci 2004;61:2924 – 31. 4. Furitsu T, Tsujimura T, Tono T, et al. Identification of mutations in the sensitivity compared with WT c-KIT and, especially, V560G coding sequence of the proto-oncogene c-kit in a human mast cell c-KIT in both cell proliferation and autophosphorylation leukemia cell line causing ligand-independent activation of c-kit product. assays. J Clin Invest 1993;92:1736 – 44. To evaluate potential second-line kinase inhibitors, we 5. Kitayama H, Kanakura Y, Furitsu T, et al. Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent tested the effects of nilotinib and PKC412 on the panel of growth and tumorigenicity of factor-dependent hematopoietic cell lines. c-KIT–expressing cell lines. Nilotinib is a phenylamino- Blood 1995;85:790 – 8.

Mol Cancer Ther 2007;6(3). March 2007

6. Kitayama H, Tsujimura T, Matsumura I, et al. Neoplastic transformation ATP pocket causes acquired resistance to imatinib in a gastrointestinal of normal hematopoietic cells by constitutively activating mutations of stromal tumor patient. Gastroenterology 2004;127:294 – 9. c-kit receptor tyrosine kinase. Blood 1996;88:995 – 1004. 30. Wakai T, Kanda T, Hirota S, Ohashi A, Shirai Y, Hatakeyama K. Late 7. Piao X, Paulson R, van der Geer P, Pawson T, Bernstein A. Oncogenic resistance to imatinib therapy in a metastatic gastrointestinal stromal mutation in the Kit receptor tyrosine kinase alters substrate specificity and tumour is associated with a second KIT mutation. Br J Cancer 2004;90: induces degradation of the protein tyrosine phosphatase SHP-1. Proc Natl 2059 – 61. Acad Sci U S A 1996;93:14665 – 9. 31. Daub H, Specht K, Ullrich A. Strategies to overcome resistance to 8. Longley BJ, Jr., Metcalfe DD, Tharp M, et al. Activating and dominant targeted protein kinase inhibitors. Nat Rev Drug Discov 2004;3:1001 – 10. inactivating c-KIT catalytic domain mutations in distinct clinical forms of 32. McLean SR, Gana-Weisz M, Hartzoulakis B, et al. Imatinib binding and human mastocytosis. Proc Natl Acad Sci U S A 1999;96:1609 – 14. cKIT inhibition is abrogated by the cKIT kinase domain I missense mutation 654 9. Ashman LK, Ferrao P, Cole SR, Cambareri AC. Effects of mutant c-kit in Val Ala. Mol Cancer Ther 2005;4:2008 – 15. early myeloid cells. Leuk Lymphoma 2000;37:233 – 43. 33. Heinrich MC, Corless CL, Blanke CD, et al. Molecular correlates of 10. Beghini A, Peterlongo P, Ripamonti CB, et al. C-kit mutations in core imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol 2006; binding factor leukemias. Blood 2000;95:726 – 7. 96:925 – 32. 11. Kemmer K, Corless CL, Fletcher JA, et al. KIT mutations are common 34. Frost MJ, Ferrao PT, Hughes TP, Ashman LK. Juxtamembrane mutant in testicular seminomas. Am J Pathol 2004;164:305 – 13. V560GKit is more sensitive to imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant. Mol Cancer 12. Reilly JT. Pathogenesis of acute myeloid leukaemia and inv(16) Ther 2002;1:1115 – 24. (p13;q22): a paradigm for understanding leukaemogenesis? Br J Haematol 2005;128:18 – 34. 35. Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 13. Wang YY, Zhou GB, Yin T, et al. AML1-ETO and C-KIT mutation/ 1993;90:8392 – 6. overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec. Proc Natl Acad Sci U S A 2005;102:1104 – 9. 36. Young SM, Cambareri AC, Ashman LK. Role of c-KIT expression level and phosphatidylinositol 3-kinase activation in survival and proliferative 14. Cammenga J, Horn S, Bergholz U, et al. Extracellular KIT receptor responses of early myeloid cells. Cell Signal 2006;18:608 – 20. mutants, commonly found in core binding factor AML, are constitutively active and respond to imatinib mesylate. Blood 2005;106:3958 – 61. 37. Mol CD, Lim KB, Sridhar V, et al. Structure of a c-kit product complex reveals the basis for kinase transactivation. J Biol Chem 2003;278: 15. Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-function mutations of 31461 – 4. c-kit in human gastrointestinal stromal tumors. Science 1998;279:577 – 80. 38. Mol CD, Dougan DR, Schneider TR, et al. Structural basis for the 16. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase. J Biol Chem stromal tumors. J Clin Oncol 2004;22:3813 – 25. 2004;279:31655 – 63. 17. Dewar AL, Cambareri AC, Zannettino AC, et al. Macrophage colony- 39. Foster R, Griffith R, Ferrao P, Ashman L. Molecular basis of the stimulating factor receptor c-fms is a novel target of imatinib. Blood 2005; constitutive activity and STI571 resistance of Asp816Val mutant KIT 105:3127 – 32. receptor tyrosine kinase. J Mol Graph Model 2004;23:139 – 52. 18. Buchdunger E, Zimmermann J, Mett H, et al. Inhibition of the Abl 40. Growney JD, Clark JJ, Adelsperger J, et al. Activation mutations of in vitro in vivo protein-tyrosine kinase and by a 2-phenylaminopyrimidine human c-KIT resistant to imatinib mesylate are sensitive to the tyrosine derivative. Cancer Res 1996;56:100 – 4. kinase inhibitor PKC412. Blood 2005;106:721 – 4. 19. Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan 41. Casteran N, De Sepulveda P, Beslu N, et al. Signal transduction by J. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. several KIT juxtamembrane domain mutations. Oncogene 2003;22: Science 2000;289:1938 – 42. 4710 – 22. 20. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective 42. Carter TA, Wodicka LM, Shah NP, et al. Inhibition of drug-resistant inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. mutants of ABL, KIT, and EGF receptor kinases. Proc Natl Acad Sci U S A Nat Med 1996;2:561 – 6. 2005;102:11011 – 6. 21. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific 43. Azam M, Latek RR, Daley GQ. Mechanisms of autoinhibition and STI- inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N 571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 2003; Engl J Med 2001;344:1031 – 7. 112:831 – 43. 22. Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. 44. Tamborini E, Pricl S, Negri T, et al. Functional analyses and molecular Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective modeling of two c-Kit mutations responsible for imatinib secondary tyrosine kinase inhibitor. Blood 2000;96:925 – 32. resistance in GIST patients. Oncogene 2006;25:6140 – 6. 23. Dagher R, Cohen M, Williams G, et al. Approval summary: imatinib 45. Heinrich MC, Corless CL, Demetri GD, et al. Kinase mutations and mesylate in the treatment of metastatic and/or unresectable malignant imatinib response in patients with metastatic gastrointestinal stromal gastrointestinal stromal tumors. Clin Cancer Res 2002;8:3034 – 8. tumor. J Clin Oncol 2003;21:4342 – 9. 24. Gorre ME, Mohammed M, Ellwood K, et al. Clinical resistance to STI- 46. Kantarjian H, Giles F, Wunderle L, et al. Nilotinib in imatinib-resistant 571 cancer therapy caused by BCR-ABL gene mutation or amplification. CML and Philadelphia chromosome-positive ALL. N Engl J Med 2006;354: Science 2001;293:876 – 80. 2542 – 51. 25. Shah NP, Nicoll JM, Nagar B, et al. Multiple BCR-ABL kinase domain 47. von Bubnoff N, Gorantla SH, Kancha RK, Lordick F, Peschel C, mutations confer polyclonal resistance to the tyrosine kinase inhibitor Duyster J. The systemic mastocytosis-specific activating cKit mutation imatinib (STI571) in chronic phase and blast crisis chronic myeloid D816V can be inhibited by the tyrosine kinase inhibitor AMN107. leukemia. Cancer Cell 2002;2:117 – 25. Leukemia 2005;19:1670 – 1. 26. Chen LL, Trent JC, Wu EF, et al. Amissense mutation in KIT kinase 48. Gleixner KV, Mayerhofer M, Aichberger KJ, et al. PKC412 inhibits domain 1 correlates with imatinib resistance in gastrointestinal stromal in vitro growth of neoplastic human mast cells expressing the D816V- tumors. Cancer Res 2004;64:5913 – 9. mutated variant of KIT: comparison with AMN107, imatinib, and cladribine 27. Debiec-Rychter M, Cools J, Dumez H, et al. Mechanisms of resistance (2CdA) and evaluation of cooperative drug effects. Blood 2006;107: to imatinib mesylate in gastrointestinal stromal tumors and activity of the 752 – 9. PKC412 inhibitor against imatinib-resistant mutants. Gastroenterology 49. Prenen H, Cools J, Mentens N, et al. Efficacy of the kinase inhibitor 2005;128:270 – 9. SU11248 against gastrointestinal stromal tumor mutants refractory to 28. Antonescu CR, Besmer P, Guo T, et al. Acquired resistance to imatinib imatinib mesylate. Clin Cancer Res 2006;12:2622 – 7. in gastrointestinal stromal tumor occurs through secondary gene mutation. 50. Gotlib J, Berube C, Growney JD, et al. Activity of the tyrosine kinase Clin Cancer Res 2005;11:4182 – 90. inhibitor PKC412 in a patient with mast cell leukemia with the D816V KIT 29. Tamborini E, Bonadiman L, Greco A, et al. A new mutation in the KIT mutation. Blood 2005;106:2865 – 70.

MolCancerTher2007;6(3).March2007

Kathryn G. Roberts, Adam F. Odell, Ellen M. Byrnes, et al.

Mol Cancer Ther 2007;6:1159-1166.

Updated version Access the most recent version of this article at: http://mct.aacrjournals.org/content/6/3/1159

Cited articles This article cites 50 articles, 28 of which you can access for free at: http://mct.aacrjournals.org/content/6/3/1159.full#ref-list-1

Citing articles This article has been cited by 13 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/6/3/1159.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/6/3/1159. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.