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[Cancer Research 65, 6631-6639, August 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

A Knock-In Mouse Model of Gastrointestinal Stromal Tumor Harboring Kit K641E

Brian P. Rubin1, Cristina R. Antonescu4, James P. Scott-Browne1, Melissa L. Comstock1, Yansong Gu2, Munir R. Tanas1, Carol B. Ware3 and Jessica Woodell1

Departments of 1 Pathology, 2 Radiation Oncology and Immunology, and 3 Comparative Medicine, University of Washington Medical Center, Seattle, Washington and 4 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York

Requests for reprints: Brian P. Rubin, Department of Anatomic Pathology, University of Washington Medical Center, 1959 Northeast Pacific Street, Box 356100, Seattle, WA 98195. Phone: 206-598-5024; Fax: 206-598-8697; E-mail: bprubin{at}u.washington.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A mouse model of gastrointestinal stromal tumor (GIST) has been developed by a knock-in gene targeting strategy, which introduced a Kit gene K641E mutation, originally identified in sporadic human GISTs and in the germ line of familial GIST syndrome patients. Homozygous and heterozygous Kit K641E mice develop gastrointestinal pathology with complete penetrance and all Kit K641E homozygotes die by age 30 weeks due to gastrointestinal obstruction by hyperplastic interstitial cells of Cajal (ICC) or GISTs. Heterozygous mice have less extensive ICC hyperplasia and smaller GISTs, suggesting a dose-response relationship between oncogenically activated Kit and ICC proliferation. Immunoprecipitation and Western blotting reveal GISTs to contain abundant phosphorylated/activated Kit. In addition to ICC hyperplasia and GISTs, homozygous Kit K641E mice exhibit loss-of-function Kit phenotypes, including white coat color, decreased numbers of dermal mast cells, and sterility, indicating that despite its oncogenic activity the mutant form cannot accomplish many activities of the wild-type gene. Kit K641E reproduces the pathology associated with the familial GIST syndrome and thus is an excellent model to study Kit pathway activation, ICC biology, GIST pathogenesis, and preclinical validations of GIST therapies and mechanisms of drug resistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The important role of receptor tyrosine kinases (RTK) in cancer has been firmly established by recent developments in both the pathogenesis and therapy of human cancer. Numerous RTKs are either mutationally activated or involved in translocations that result in constitutive, ligand-independent kinase activity (1). A breakthrough in therapy for cancers associated with activating mutations in tyrosine kinases was the development of imatinib mesylate (Glivec, Gleevec) for the treatment of chronic myelogenous leukemia (CML; ref. 2). Imatinib targets BCR-Abl kinase in CML, resulting in strong inhibition of BCR-Abl-mediated signaling. IFN-refractory CML responds dramatically to imatinib; this development of a molecularly targeted therapy is a landmark in cancer treatment. Imatinib was subsequently shown to inhibit Kit, platelet-derived growth factor receptors (PDGFR) A and B, and some neoplasms driven by activating mutations in these RTKs, also respond to imatinib therapy.

Kit is a RTK with a split intracellular kinase domain and is related structurally to colony-stimulating factor-1 receptor (also known as FMS receptor), Flt-3 receptor, and PDGFRA and PDGFRB (3). Kit ligand (also known as steel factor, stem cell factor, and mast cell growth factor) is the only known ligand for Kit (4). Kit, located on chromosome 4q in humans, was originally discovered as a viral proto-oncogene (v-Kit) that caused sarcomas in cats, and its role as a cellular oncogene has been further established in acute myelogenous leukemia, mast cell leukemia, mastocytosis, germ cell tumors, and gastrointestinal stromal tumors (GIST; refs. 5–11). Kit mutations within these neoplasms are defined as "activating" because they confer ligand-independent growth when transfected into otherwise Kit ligand-dependent Ba/F3 cells in vitro and tumors when transfected cells are injected into nude mice (5, 12). Kit is involved in the development and maintenance of hematopoietic stem cells, mast cells, melanocytes, germ cells, and interstitial cells of Cajal (ICC; refs. 5, 13–16). Loss-of-function causes developmental defects in all of these cellular compartments, including anemia, sterility, loss of ICC, and white coat color "white spotting" due to a failure of melanocytes to migrate during melanogenesis (4, 13, 17, 18).

GISTs are believed to arise from the ICC lineage (5, 19). ICC are mesenchymal cells located within the muscular wall of the gastrointestinal tract, where they coordinate peristalsis through inherent pacemaker function (13, 18, 20, 21). There is evidence to suggest that both ICC and smooth muscle of the gut wall arise from a common stem cell and that although Kit expression is not necessary for fate determination Kit is essential for maintenance and proliferation of ICCs (17). Indeed, Kit antigen is the immunohistochemical marker of choice for identifying ICCs (5, 17–19). Loss-of-function Kit mutations and/or decreased Kit levels not only result in ICC hypoplasia but also have functional consequences as shown by disturbances in peristalsis and, in extreme cases, severe dysmotility (13, 22).

As a mesenchymal derivative, GISTs are sarcomas; the most common sarcoma of the gastrointestinal tract. At least two signal transduction pathways are important in the pathogenesis of GISTs; activating mutations in Kit and PDGFRA have been identified in 88% and 5% of GISTs, respectively (5, 12, 23). Additional support for the relationship between mutational activation of Kit or PDGFRA and pathogenesis of GIST came from studies of familial GIST, which is characterized by ICC hyperplasia and multiple GISTs with an autosomal dominant pattern of inheritance (10, 24–28). Familial GIST patients carry activating germ line Kit or PDGFRA mutations identical to those found in sporadic GISTs.

Thus, the accumulated evidence argues strongly for a role for Kit in the development of ICCs and their transformation into GISTs. Unlike the multistep pathways elucidated for colon cancer, little is known about the intervening steps in the genesis of sarcomas in general or GIST in particular. Therefore, we sought to create a mouse model of GIST by introducing an activating Kit mutation, K641E, associated with human familial GIST syndrome (K642E in human Kit) into the mouse genome (27). This mouse model was anticipated to provide unique insights into the role of Kit in the pathogenesis of GISTs as well as the biology of ICCs and normal Kit function. Furthermore, it was apparent that such a model would be useful in the preclinical evaluation of potential anti-Kit therapies and the study of mechanisms of resistance to these therapies. Herein, we report a "knock-in" transgenic mouse model with activated oncogenic Kit expression, impressive ICC hyperplasia, GISTs, and, surprisingly, multiple manifestations of decreased Kit function. This mouse model is not without precedent, as another group recently reported the construction of a similar mouse model harboring a Kit V558 deletion (29).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of mutant KitK641E mice. Site-directed mutagenesis using QuickChange XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and primer A introduced a K-to-E mutation at amino acid position 641 within Kit exon 13. The mutation also introduced a novel XhoI restriction site. A 4.9-kb HindIII-HindIII fragment spanning exons 8 to 13 and containing the K641E mutation and an adjacent 2.1-kb HindIII-EcoRI fragment containing exon 14 were subcloned on either side of a neomycin resistance gene under the control of the W/UV5 bacterial promoter in the same orientation as the Kit gene, flanked by loxP sites within targeting vector 4317G9 (provided by Dr. Richard Palmiter, University of Washington, Seattle, WA). This targeting vector (described in Fig. 1A) also featured two negative selection cassettes on either side of the cloned fragments: the diphtheria toxin A gene (DT-A) and the herpes simplex virus-thymidine kinase gene (HSV-TK). A unique BglII restriction site within a polylinker 5' of the neomycin resistance cassette was also present and was used in mapping homologous integration by Southern blotting. 129Sv embryonic stem cells were electroporated with AscI linearized targeting construct using standard protocols. Ninety-four neomycin-resistant clones were isolated and analyzed for homologous integration by Southern blot analysis after BglII digestion with a 3' Kit probe, which flanked the region of the Kit gene used in the targeting construct. The presence of the K641E mutation was confirmed by amplification with primers B and C of a 478-bp fragment harboring the mutation and a novel XhoI site that was introduced by the exon 13 mutation. XhoI digestion of this fragment yields 403- and 75-bp fragments. The sequence of the PCR fragments was confirmed by DNA sequence analysis. Two correctly targeted clones were identified and one of these clones was microinjected into C57B6 blastocysts and transferred to the uterus of E2.5 pseudopregnant recipients according to standard procedures (E0.5 is the day of plug). Male offspring displaying at least 50% chimerism were backcrossed to C57B6 and 129SvJ females, respectively, to establish germ line transmission. Mutant mice were maintained on a mixed (129Sv x C57B6) or 129Sv genetic background as described. All mouse experiments were done with approval of the University of Washington Animal Care and Use Committee and in accordance with the guidelines established by the NIH. The primers are as follows: primer A, 5'-GCCCTAATGTCGGAACTCGAGGTCCTGAGCTACCTG-3'; primer B, 5'-AGTTGGCAGGGTTAGCAGAA-3'; and primer C, 5'-AGACTCACCTCCCACCGTGC-3'.



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  		Figure 1. Targeting strategy. A, schematic representation of K641E targeting strategy. Restriction enzyme sites are as follows: B, BglII; H, HindIII; E, EcoRI. Negative selection was achieved through DT-A at the 5' end of the construct and HSV-TK at the 3' end of the construct. Positive selection was achieved through a neomycin resistance cassette (neo). Location of 3' primer used for Southern blots and PCR primers are shown. B, PCR genotyping assay containing representative Kit+/+, Kit+/K641E:Neo, and KitK641E:Neo/K641E:Neo tail DNAs. The first lane of each pair shows the results of digestion with XhoI; the K641E mutation introduced a novel XhoI site into the PCR product resulting in a 403-bp product after digestion with XhoI. D, XhoI digested PCR products; U, undigested PCR products.

 
Flow cytometry. Single-cell suspensions of bone marrow from 6- to 12-week-old littermate mice were prepared from femur and tibia. RBCs were lysed with ACK lysis buffer and nonspecific staining was blocked using anti-Fc receptor 24G2 hybridoma supernatant. Cells were stained on ice in PBS containing 2% FCS and 0.05% sodium azide at 2 x 107/mL with allophycocyanin-conjugated CD117 (Kit) or isotype control (rat IgG2b, {kappa}) and phycoerythrin-conjugated CD90.2 (Thy1.2), CD11b (Mac-1), Ly6G (Gr-1), CD45R (B220), and TER119 (BD Biosciences, San Jose, CA). Samples were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software (Treestar).

Immunoprecipitation and Western blotting. A representative portion of all GISTs was snap frozen for biochemical analysis. Snap-frozen tumor (0.04 g) was ground to powder in liquid nitrogen, resuspended in NP40 lysis buffer (1 mL) containing 50 mmol/L Tris-HCL (pH 8.0), 150 mmol/L NaCl, 5 mmol/L EDTA, 1% NP40, and mini-tablet protease mix (1 tablet/10 mL, Roche Applied Science, Indianapolis, IN), passed through a 21-gauge needle 10 times, incubated on ice for 30 minutes, and centrifuged at 16,000 x g (14,000 rpm in an Eppendorf 5415C centrifuge) for 10 minutes at 4°C. Lysates were precleared with 20 µL protein G-Sepharose beads (Zymed Laboratories, San Francisco, CA) for 30 minutes. Precleared lysates were precipitated for 1 hour with goat anti-mouse anti-Kit antibody (M-14, Santa Cruz Biotechnology, Santa Cruz, CA) followed by 1-hour incubation with 20 µL protein G-Sepharose at 4°C. Immunoprecipitates were washed and fractionated on 8% PAGE containing 0.1% SDS. For Western blotting, rabbit anti-Kit (C-19, Santa Cruz Biotechnology) or mouse anti-Tyr(P) (PY99, Santa Cruz Biotechnology) were used. Secondary antibodies were either horseradish peroxidase–conjugated goat anti-rabbit or anti-mouse IgG (Zymed Laboratories). The blots were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL).

Histologic analysis and immunohistochemistry. Complete necropsies were done on three Kit+/K641E:Neo and three KitK641E:Neo/K641E:Neo mice. Organs examined included brain, eyes, heart, lungs, thymus, liver, spleen, gastrointestinal tract, kidneys, bladder, gonads, bone marrow, and skin. Because the gastrointestinal tract, gonads, and skin were abnormal, these tissues were routinely harvested from a large cohort of Kit+/+ (n = 11), Kit+/K641E:Neo (n = 22), and KitK641E:Neo/K641E:Neo (n = 35) mice. Sections (4 µm) from tissues fixed in 10% formalin were stained with H&E or used for immunohistochemistry. Immunohistochemistry was done by the avidin-biotin-peroxidase complex technique using commercially available antibodies to the following antigens: CD34 (QBEnd 10; 1:100; DAKO, Carpinteria, CA), Kit (polyclonal, 1:150, Santa Cruz Biotechnology), {alpha}-smooth muscle actin (1A4, 1:2,000, Beckman Coulter, Fullerton, CA), desmin (D33, 1:500, DAKO), and inhibin (inhibin {alpha} monoclonal, 1:10, Serotec, Raleigh, NC). Antigen retrieval consisted of 15 minutes of microwave pretreatment in citrate buffer before the application of all primary antibodies. Appropriate positive and negative controls were done.

Electron microscopy. Tissue was fixed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in epoxy resin using standard procedures. Sections were examined with a Philips EM410 transmission electron microscope (Philips, Eindhoven, the Netherlands).

Determination of mast cell numbers. Skin mast cell numbers were determined by counting the number of mast cells within 10 x400 fields (Olympus BX51 microscope, Olympus America, Melville, NY) in sections from the dermis of the back after staining with 1% toluidine blue for 2 minutes. Sections were counted twice by each of two pathologists (B.P.R. and M.T.) and the numbers were averaged.

Statistical analysis. Mendelian frequencies were evaluated by {chi}2 analysis. To evaluate differences in survival between different genotypes, a log-rank test was done. The data are presented via Kaplan-Meier curves. To evaluate differences in the number of dermal mast cells, ANOVA and Tukey's tests were done. To evaluate differences in cecal diameter, a square root transformation was done and the data were evaluated by one-way ANOVA and the Bonferroni multiple comparisons test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Introduction of KitK641E into the mouse genome by allelic conversion. Lys641 is located within the first portion of the split kinase domain encoded by exon 13, which shows 97% identity at the protein level between human and mouse Kit proteins (30, 31). Overall, mouse and human Kit are 82% identical at the protein level. By analogy to familial GIST patients, we reasoned that mice harboring a Kit K641E allele would also develop ICC hyperplasia and GISTs. To model GISTs in mice, we developed a targeting vector to introduce the Kit K641E mutation, which causes familial GISTs in humans, into the mouse Kit locus. The targeting vector consisted of a 7-kb fragment of the Kit gene containing exons 8 to 14, including the K641E mutation, which was introduced by a site-directed mutagenesis strategy, and a neomycin resistance cassette flanked by loxP sites within intron 13 (Fig. 1A). Homologous integration of the targeting vector into 129Sv embryonic stem cells resulted in two correctly targeted embryonic stem cell clones, which were confirmed by Southern blot hybridization, PCR, and DNA sequence analysis. Embryonic stem cells from one of two clones were microinjected into C57B6 blastocysts, and the resultant male chimeras were backcrossed with C57B6 females to establish germ line transmission, which was confirmed by Southern blot hybridization and PCR (Fig. 1B).

Loss-of-function phenotypes in KitK641E mice. Breeding of Kit+/K641E:Neo mice produced 243 offspring of the following genotypes: 125 Kit+/+, 78 Kit+/K641E:Neo, and 40 KitK641E:Neo/K641E:Neo mice. There is a clear departure from Mendelian distribution ({chi}2 analysis, {chi}2 = 90.61, P < 0.001; 95% confidence limits). Whereas Kit+/K641E:Neo mice were normal externally with black coats, KitK641E:Neo/K641E:Neo mice had uniform white coats (Fig. 2A), which suggested that the mutant KitK641E:Neo allele phenocopied the "white spotting" phenotype, previously associated with loss of Kit function. Unlike albino mice, which have pink eyes, these "white spotting" mice have normally pigmented dark brown eyes consistent with a specific defect. Flow cytometric analysis of adult bone marrow from homozygous mutants revealed an undetectable level of cell surface Kit expression in contrast with heterozygous or wild-type littermates (Fig. 2B). In conjunction with this decreased KitK641E:Neo expression, the striking white phenotype strongly suggests loss of Kit function mechanism in this mouse model. Furthermore, homozygous KitK641E:Neo/K641E:Neo mice displayed two other loss-of-function phenotypes: low numbers of dermal mast cells and infertility; both males and females failed to produce offspring when bred with Kit+/+ littermates. The latter two phenotypes were investigated histologically.



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  		Figure 2. Loss-of-function Kit phenotypes. A, homozygous KitK641E:Neo/K641E:Neo "white spotting" mouse with uniformly white coat and black eyes. B, flow cytometric analysis of bone marrow. The distribution of Kit expression versus a panel of lineage (Lin) markers (Thy1.2, CD11b, G4-1, B220, and TER119) is shown. Expression of Kit is high on Lin– in Kit+/+ mice (long arrow, top), ~1% to 2% of total bone marrow, and intermediate on a subset of Linhi cells (short arrow, top). In KitK641E:Neo/K641E:Neomice, Kit expression is undetectable over the isotype control antibody for Linhi subsets and reduced >10-fold in the Lin– population. In heterozygous Kit+/K641E:Neo mice, Kit expression is reduced slightly in Linhi and Lin– compared with Kit+/+ mice. C, 1-month-old KitK641E:Neo/K641E:Neo testis with seminiferous tubules showing a single layer of germ cells and a complete lack of spermatozoa (arrow). D, 3-month-old testis showing extensive Leydig cell hyperplasia (*) and germ cells that are sloughed into the center of the seminiferous tubules (arrow). E, 7-month-old KitK641E:Neo/K641E:Neo ovary composed only of spindle-shaped and luteinized stromal cells (short arrow); germ cells are not identified. Unusual tubular structures are identified (long arrow). F, decreased numbers of dermal mast cells in KitK641E:Neo/K641E:Neomice compared with either Kit+/K641E:Neo or Kit+/+ mice (P < 0.001).

 
Microscopically, the reason for the sterility phenotype in KitK641E:Neo/K641E:Neo mice was obvious. Male KitK641E:Neo/K641E:Neo mice did not contain mature spermatids and female KitK641E:Neo/K641E:Neo mice lacked identifiable germ cells. The testes contained seminiferous tubules with only one layer of primary germ cells and a complete absence of spermatids and spermatozoa (Fig. 2C). Instead, the seminiferous tubules contained abundant, pale, eosinophilic, flocculent secretions (Fig. 2C). As the KitK641E:Neo/K641E:Neo mice increased in age, there was progressive Leydig cell hyperplasia, and by age 3 months, the testis had severe Leydig cell hyperplasia and the germ cells were sloughed into the center of the seminiferous tubules (Fig. 2D). The ovaries of the KitK641E:Neo/K641E:Neo females were small, difficult to find, and devoid of germ cells (Fig. 2E). They consisted of stromal cells that ranged from spindle shaped to more rounded inhibin-positive/Kit-negative luteinized cells (data not shown). In some of the ovaries, the stromal cells were arranged focally in tubules (Fig. 2E). The gonads of Kit+/K641E:Neo mice were histologically normal.

Because the density of dermal mast cells is dependent on Kit levels, we postulated that we might observe changes in the density or distribution of these cells as a functional consequence of activated Kit expression. We examined sections of skin and determined the relative numbers of mast cells as a function of genotype. The number of mast cells in the KitK641E:Neo/K641E:Neo mice was lower than in either heterozygous Kit+/K641E:Neo or homozygous Kit+/+ mice; KitK641E:Neo/K641E:Neo = 0.0 to 14.5 (median, 3.5; n = 18), Kit+/K64E:Neo = 20.0 to 145.0 (median, 45.8; n = 16), and Kit+/+ = 29.5 to 52.5 (median, 43; n = 7; P < 0.001; Fig. 2F). Thus, to our surprise, the KitK641E:Neo/K641E:Neo mice exhibited three different loss-of-function phenotypes: white coat color, sterility, and decreased density of dermal mast cells. However, as described below, this same mouse exhibited a striking gain-of-function phenotype within the gastrointestinal tract.

Interstitial cells of Cajal hyperplasia and gastrointestinal stromal tumors in KitK641E:Neo mice. Within 3 weeks of birth, coincident with weaning and the onset of solid food intake, a subset of KitK641E:Neo/K641E:Neo mice developed distended abdomens and died within days of apparent intestinal obstruction. A protocol was instituted to sacrifice all mice as soon as they began to exhibit symptoms of abdominal distension, so that necropsies could be done and tissue could be collected, before the occurrence of significant autolysis. KitK641E:Neo/K641E:Neo mice developed symptoms and either died or were sacrificed due to symptoms of obstruction from 3 to 30 weeks (median, 5 weeks; P < 0.001; Fig. 3A), whereas their Kit+/K641E:Neo littermates were viable and asymptomatic.



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  		Figure 3. A, Kaplan-Meier curve of Kit+/+, Kit+/K641E:Neo, and KitK641E:Neo/K641E:Neo mice. Survival is diminished significantly in KitK641E:Neo/K641E:Neo mice, with all of these mice succumbing to gastric or intestinal obstruction by age 30 weeks (P < 0.001). B, normal appearance of the abdominal contents of a 1-month-old Kit+/+ mouse compared with the contents of a 1-month-old KitK641E:Neo/K641E:Neo mouse with mega-ileum due to a large cecal GIST (C). The arrow in B points to the normal small bowel. All that can be seen within the bulging abdomen of the mouse in C is the massively distended small bowel filled with fecal material. D, gastrointestinal tract of a 6-month-old KitK641E:Neo/K641E:Neo mouse with an enlarged and grossly distended stomach (arrowhead) and cecum involved by a massive GIST measuring 1.2 cm in greatest diameter (arrow). E, close-up of the cecum from D. The cecum is involved by a massive GIST, which is white in contrast to the normal color of the cecum (brown). F, cross-sections of GIST showing fleshy cut surface and remaining cecal lumen (arrow). G, expression and phosphorylation of Kit in GISTs from two different KitK641E:Neo/K641E:Neo mice (#1 and #2). Protein lysates were immunoprecipitated with anti-Kit antibody M-14 and analyzed by SDS-PAGE. Membranes were blotted with anti-Kit antibody C-19 (top) and anti-phosphotyrosine antibody PY99 (bottom). A protein lysate from mouse P815 mastocytoma cell line [M; courtesy of Dr. Michael Heinrich (Oregon Health Sciences University, Portland, OR)] is shown as a control. Note that the GISTs contain considerably more activated Kit than the mastocytoma cell line that was shown to be phosphorylated at longer exposures.

 
Because we hypothesized that the Kit K641E mutation would cause ICC hyperplasia and GIST formation, a detailed examination of the gastrointestinal tract was done in each mouse. Grossly, the gastrointestinal tract of KitK641E:Neo/K641E:Neo mice was obstructed at the pylorus (22 of 35 mice) or the cecum (12 of 35 mice), leading to a distended stomach or mega-ileum, respectively (Figs. 3B-D and 4A). Furthermore, the ceca of homozygous KitK641E:Neo/K641E:Neo mice were always abnormally firm and contained tumors that could be distinguished grossly (Fig. 3D-F). Ceca of the differing genotypes had the following diameters: KitK641E:Neo/K641E:Neo = 0.05 to 1.2 cm (average, 0.19 cm; n = 35), Kit+/K64E:Neo = 0.03 to 0.22 cm (average, 0.11 cm; n = 18), and Kit+/+ mice = 0.01 to 0.02 cm (average, 0.015 cm; n = 10). The difference in cecum diameter between Kit+/K641E:Neo or KitK641E:Neo/K641E:Neo and Kit+/+ mice was statistically significant (P < 0.001). The difference between Kit+/K641E:Neo and KitK641E:Neo/K641E:Neo mice was also statistically significant (P = 0.05). Thus, expression of activated Kit has an allele dosage–dependent, profound effect on the gastrointestinal tract, culminating in frank intestinal obstruction and death.



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  		Figure 4. ICC hyperplasia in KitK641E:Neo/K641E:Neo mice. A, stomach from KitK641E:Neo/K641E:Neo mouse with extensive ICC hyperplasia leading to tensely distended stomach. Arrow, pyloric region that has a faint white color due to extensive involvement by ICC hyperplasia. The proximal third of the stomach (*) is usually paler than the rest of the stomach due to the presence of squamous mucosa, which is a normal finding. B, stomach wall from a Kit+/+ mouse. Right bracket, normal thickness of the muscular wall. C, gastric wall from a KitK641E:Neo/K641E:Neo mouse showing moderate ICC hyperplasia involving the outer half of the muscularis propria (right bracket). Note that the wall thickness is approximately twice as thick the normal stomach wall in A. D, ICC hyperplasia from a KitK641E:Neo/K641E:Neo mouse involving the muscular wall of the pylorus (right bracket). Note that hyperplasia is arising in between the muscular layers (*) from the location of Auerbach's plexus. E, low-power view of the pylorus from a KitK641E:Neo/K641E:Neo mouse involved by extensive ICC hyperplasia (right bracket), virtually replacing the outer portion of the muscularis. F, pylorus from a KitK641E:Neo/K641E:Neo mouse that is extensively infiltrated by ICC hyperplasia (*) resulting in near total obstruction of the lumen (arrow). This section was taken from a mouse that presented with clinical gastric obstruction.

 
To determine the extent of Kit expression and activation in the cecal tumors, Kit immunoprecipitation was done on tumor lysates, and the immunoprecipitate was Western blotted with anti-Kit and anti-phosphotyrosine antibodies after PAGE (Materials and Methods). The cecal masses contained substantial amounts of phosphorylated Kit (Fig. 3G). Interestingly, Kit seemed to be more strongly phosphorylated in comparison with immunoprecipitates from mouse P815 mastocytoma cell line (Fig. 3G).

To more fully understand the reasons for the obstruction phenotypes seen in the KitK641E:Neo/K641E:Neo mice, histologic examination of the entire gastrointestinal tract was done and compared with Kit+/K641E:Neo and Kit+/+ mice. This analysis revealed relatively uniform histologic findings across all mice of each genotype. Beginning within the distal esophagus and extending throughout the stomach and into the proximal duodenum, the wall of the gut of KitK641E:Neo/K641E:Neo mice was involved by ICC hyperplasia that arose from the myenteric plexus and invaded the surrounding musculature (Fig. 4C-F). ICC hyperplasia was most prominent in the pylorus, where it completely effaced the muscular wall and extended through the muscularis mucosae to engulf the mucosa, almost certainly explaining the obstruction and gastric bloating that had been seen (Fig. 4D-F). With the exception of the most proximal portion of the duodenum, the small intestine did not contain any evidence of ICC hyperplasia. In the KitK641E:Neo/K641E:Neo mice that presented with mega-ileum (n = 12 of 35), the ceca were grossly enlarged and on cross-section had lumens that were narrowed by fleshy-appearing GISTs. Histologic analysis of the GISTs revealed that they were composed of a dense, cellular proliferation of monomorphic spindle cells, similar in appearance to, but much more extensive, than the ICC hyperplasia of the upper gastrointestinal tract (Fig. 5A and C). Based on the analysis of 35 ceca from mice of various ages, the natural history of GISTs in this location seemed to follow an orderly progression (data not shown). ICC hyperplasia was first apparent within the myenteric plexus and grew asymmetrically, involving predominantly one side of the cecum. As this process continued, circumferential involvement of the gut wall resulted. At its greatest extent, the muscular wall was completely replaced by the mass that variably crowded the lumen, causing obstruction and death. Some GISTs seemed to grow outward and did not result in intestinal obstruction. The largest GISTs extended from the serosal surface, through the muscularis propria, into and through the muscularis mucosae to invade into the mucosal/epithelial surface (Fig. 5A). The colon and rectum were also circumferentially involved by diffuse ICC hyperplasia emanating from the myenteric plexus, involving, and in some areas completely replacing, the surrounding muscularis propria (data not shown). Metastases were never seen either grossly or microscopically.



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  		Figure 5. GISTs in KitK641E:Neo/K641E:Neo mice. A, very low power view of a large cecal GIST from a KitK641E:Neo/K641E:Neo mouse. Note the asymmetrical involvement as the lumen (*) is pushed to the left. B, the cecal wall from a Kit+/+ mouse is shown in the inset for comparison at the same magnification as the cecal GIST in A. A high-power view of the cecal wall is shown to highlight the lack of cellularity of the muscular wall (right bracket). C, very high power view of a cecal GIST from a KitK641E:Neo/K641E:Neo mouse showing the characteristic cytologic features: a proliferation of monomorphic, plump, spindle-shaped cells with pale fibrillary cytoplasm arranged in fascicles. Inset, a different case that had extensive paranuclear vacuolization, a finding that is commonly seen in gastric GISTs in humans. D, GIST showing diffuse and strong immunoreactivity for Kit. E, desmin-negative GIST infiltrating through the smooth muscle wall of the cecum, which is an excellent marker of smooth muscle and is seen in this panel as scattered desmin-positive (brown) cells and more extensive immunoreactivity in the portion of the muscular wall that is not involved by GIST. F, electron microscopy of cecal GIST showing undifferentiated plump spindle cells with numerous mitochondria (*). Magnification, x4,400.

 
Although heterozygous Kit+/K641E:Neo mice did not succumb to gastrointestinal lesions, necropsy revealed small cecal GISTs, and histologic analysis revealed ICC hyperplasia, similar in appearance to the ICC hyperplasia seen in KitK641E:Neo/K641E:Neo mice. However, in contrast to the homozygous mutant mice, ICC hyperplasia in the heterozygous mice involved only the cecum and large bowel and was much less extensive, showing a correlation with copy number of the activated Kit allele (data not shown). A summary of the histologic findings is presented in Table 1.


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  		Table 1. Summary of pathologic findings within the gastrointestinal tract of Kit+/+, Kit+/K641E:Neo, and KitK641E:Neo/K641E:Neo mice

 
Cytologically, hyperplasia and GISTs from Kit+/K641E:Neo and KitK641E:Neo/K641E:Neo mice were indistinguishable. The hyperplastic ICCs within the upper gastrointestinal tract were longer than those within regions of ICC hyperplasia and GISTs of the lower gastrointestinal tract (data not shown). However, overall, the morphology of the lesions was similar. The cells within ICC hyperplasia and GISTs were monomorphic, plump spindle cells with abundant pale eosinophilic, fibrillary cytoplasm and elongated nuclei with fine chromatin and inconspicuous nucleoli (Figs. 4F and 5C). Occasional cells contained paranuclear vacuoles identical to those seen in human GISTs (Fig. 5C, inset). Mitotic figures were extremely uncommon and numbered no more than 1 per 50 high-power fields (x400 field). Necrosis was not identified.

Immunohistochemistry revealed the cells regardless of whether they arose in ICC hyperplasia or GIST, or in either Kit+/K641E:Neo or KitK641E:Neo/K641E:Neo mice, to be diffusely and strongly positive for Kit (Fig. 5D) and negative for desmin (Fig. 5E), {alpha}-smooth muscle actin, CD34, and S-100 protein (data not shown).

Ultrastructural analysis was done on two cases of ICC hyperplasia involving the pylorus and two cecal GISTs. The cells exhibited similar ultrastructural findings (Fig. 5F): a spindle cell cytomorphology with smooth-contoured, fusiform nuclei containing evenly dispersed chromatin and a moderate amount of cytoplasm packed with mitochondria. A well-defined Golgi apparatus was seen in most cells. The cells within the ICC hyperplasia from the pylorus possessed short cell processes, subplasmalemmal vacuoles, and rudimentary cell junctions. In one of the two GISTs that were studied, rare cells showed focal densities, possibly representing actin microfilaments. The other GIST contained numerous perinuclear vacuoles, similar to what is seen in human GISTs. Basal lamina was not identified with the exception of an incomplete basal lamina within one of the ICC hyperplasias.

Taken together, the histologic, immunohistochemical, and ultrastructural features support the interpretation that the cells identified within the gastrointestinal tract of mice harboring either one or two copies of the KitK641E:Neo allele are hyperplastic ICCs and GISTs.

Other organs that were examined histologically were found to be normal in both Kit+/K641E:Neo and KitK641E:Neo/K641E:Neo mice (see Materials and Methods).

To exclude any influences on strain background, male chimeras were independently backcrossed to 129SvJ females to produce a 129 strain harboring Kit K641E alleles. We examined 12 KitK641E:Neo/K641E:Neo and 12 Kit+/K641E:Neo mice and found that both loss-of-function (white coat, mast cell deficiency, and sterility) and gain-of-function (ICC proliferations/GISTs) phenotypes were identical to those seen in the 129B6 mixed background (data not shown), suggesting that the genetic background does not affect the development of ICC hyperplasia or GISTs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We describe a knock-in mouse model in which a mutation found in sporadic and familial human GISTs, Kit K641E, was placed within the context of the mouse Kit gene through homologous recombination. Mice that harbored either one or two copies of the Kit K641E allele developed ICC hyperplasia and GISTs. Interestingly, the distribution of GISTs is different than is seen in humans. Sporadic human GISTs are most common in the stomach (50-60%) followed by the proximal small intestine (20-30%). Human GISTs of the large bowel are very rare (32). Human patients who carry germ line Kit K642E mutations develop both gastric and small bowel GISTs (33). Although ICC hyperplasia was identified within the same spectrum of anatomic locations as human GISTs, mouse GISTs were located within the cecum. This distribution is the same as that found within another recently described mouse GIST model in which the Kit V558{Delta} mutation (a mutation found in a familial human GIST syndrome family) was "knocked" into the Kit locus (29). The reason for the differences in distribution of GISTs between mice and humans is not clear but may have to do with differences in the density of ICC progenitor cells in different species. Nevertheless, ICC hyperplasia and GISTs identified within the mice are identical to human GISTs at all of the histologic, immunohistochemical, ultrastructural, and molecular levels and thus recapitulate many aspects of human GIST biology. As such, our model may help understand various types of developmental and acquired gastrointestinal diseases related to ICC, given that ICC hyperplasia invariably accompanied expression of the Kit K641E allele (34).

One of the fascinating and unexpected aspects of our mouse model is the apparent dose dependence of Kit activation on the extent of ICC hyperplasia and GIST formation. Mice harboring one copy of the Kit K641E allele had ICC hyperplasia limited to the colon and smaller GISTs than mice harboring two copies of the Kit K641E allele (P < 0.001). The mechanism of Kit K641E activation is unknown and it is also not known whether Kit K641E has altered substrate specificity, which might contribute to differences that are seen between Kit K641E heterozygotes and homozygotes. We expect that further overexpression of constitutively activated Kit might result in an even more pronounced neoplastic phenotype. Because ICC hyperplasia was not seen in the stomachs of Kit+/K641E:Neo mice, although it was seen in the stomachs of KitK641E:Neo/K641E:Neo mice, it suggests that gastric ICCs may have a higher threshold for response to activated Kit than colonic ICCs.

Although the gain-of-function phenotypes that were seen in the Kit K641E mice were expected, the finding of three loss-of-function phenotypes in these mice was unanticipated. The loss-of-function phenotypes could be due to impaired expression of the KitK641E:Neo allele retaining the Neo cassette. Poor expression has been seen in other genetically targeted Kit alleles harboring a neomycin resistance cassette within Kit introns (35). We are currently in the process of excising the neomycin resistance cassette in intron 13 by Cre-mediated recombination of the loxP sites, which flank this cassette, to see if this affects Kit expression or has phenotypic consequences.

The presence of both loss-of-function and gain-of-function phenotypes begs the question as to why ICC lineage cells are activated by the Kit K641E allele, whereas Kit-dependent mast cells, melanocytes, and germ cells are not. Does the ICC lineage express particularly high levels of the oncogenic Kit allele or does something inherent in the Kit K641E mutation and/or Kit signaling pathway permit a dramatic response to low levels of the mutant receptor? It is known that mouse and human mast cell leukemias/mastocytosis and human germ cell neoplasms possess activating Kit mutations, but typically, these mutations are not within the same region of the Kit gene as those seen in GISTs (6, 36). Kit mutations found in GISTs most commonly involve the intracellular juxtamembrane domain in exon 11, whereas mast cell and germ cell neoplasms generally have mutations in the phosphotransferase domain in exon 17. In addition, some Kit alleles are associated with more widespread effects, including gain-of-function phenotypes in cellular lineages other than ICC. For example, the mouse model developed by Sommer et al. not only produces ICC hyperplasia and GISTs but also develop unusual proliferations of neuroectodermal cells with features of melanocytes/melanotic Schwann cells and increased (four times) numbers of dermal mast cells (29). Their phenotype is not a total surprise because the affected individuals from the familial GIST syndrome family that this mouse is based on also have urticaria (increased dermal mast cells) in addition to ICC hyperplasia and GISTs (10). Thus, although activating Kit alleles can be selective in terms of which cell lineages are activated, not all Kit alleles exhibit such exquisite selectivity.

It is likely that differences in proliferative capacity/activation in response to different mutations lie in inherent differences in the cell types in which Kit is expressed. At the molecular level, this leads to the hypothesis of lineage-specific proteins that interact with Kit. Kit is known to act directly and indirectly with many different proteins, and the juxtamembrane domain, the site of the majority of Kit mutations found in GISTs, has key roles in protein-protein interactions (37). Although the explanation for the exquisite lineage specificity seen in our model remains unexplained, the power of mouse genetics and the availability of relatively homogeneous lesions for study should allow us to explore this question more thoroughly.

Based on the identification of Kit mutations in the majority of sporadic and familial human GISTs as well as the dramatic ICC hyperplasia and GIST phenotypes in the mice described in this report, it is clear that Kit activation is sufficient to initiate GIST formation. ICC proliferation is an important early event that dramatically increases the number of cellular targets for accrual of further genetic changes that are instrumental in neoplastic progression to malignant/metastatic phenotypes in GIST. Furthermore, activating Kit mutations are identified within human GISTs that are discovered incidentally and measure <1 cm in greatest dimension, which also supports the idea that Kit activation is the first step in the genesis of GISTs (38). Metastatic neoplasms have not been identified in the mice described in this article thus far or in a similar knock-in mouse GIST model that was published recently (29). We believe that through the considerable power of mouse genetics it will be possible to tease apart the pathway of GIST neoplastic progression.

After the discovery that Kit was mutationally activated in human GISTs, it was proposed that targeted anti-Kit therapies might be useful in their treatment (39, 40). This has turned out to be the case as initial clinical trials with imatinib, a potent small molecule inhibitor of Kit, have shown considerable promise (12, 41). As has been seen in imatinib therapy of CML, some patients who have been maintained on imatinib for many months have developed resistance to the drug. Although very little data are available at this time, the most common mechanism of resistance in GIST is through acquisition of a secondary mutation in the imatinib-resistant phosphotransferase domain encoded by exon 17 (42, 43). The mouse model described in this article may be useful in modeling resistance to different anti-Kit therapies and serve as an excellent preclinical model for the testing of anti-Kit/anti-GIST therapies that are developed in the future.

In this report, we have described a novel knock-in mouse model, whereby Kit K641E, a mutation that was originally identified in sporadic and familial human GISTs, was placed within the mouse Kit gene through homologous recombination. Mice with this mutation exhibit widespread ICC hyperplasia and GISTs and loss-of-function phenotypes affecting melanocytes, mast cells, and germ cells. The complex phenotypes exhibited by these mice highlight the intricacies of Kit signal transduction within different cellular lineages. It is hoped that this model will be useful in understanding various aspects of Kit signal transduction, the biology of ICCs, and the pathogenesis of GIST, especially the mechanism of tumor progression in GIST. Furthermore, the model has great potential as a preclinical system for testing targeted therapies of the Kit pathway as well as resistance to anti-Kit–based therapies. Finally, as Kit activation in GIST has become a paradigm for activated RTKs in sarcoma, it is hoped that this mouse model may also shed light on the pathogenesis and potential targets for treatment of related, as yet untreatable, sarcomas.


   Acknowledgments
 
Grant support: University of Washington Royalty Research Fund (B.P. Rubin DF).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Yuki Kimura and Alan Bernstein for Kit clone 3a12a, which was used to make the targeting construct; Dr. Richard Palmiter for advice on designing gene targeting vectors and vector 4317G9, which served as the backbone of our targeting vector; Dr. Brent Wood for advice on flow analysis; Dr. Michael Heinrich for the P815 mastocytoma cell line; Monica Chaudari, Eric Johnson, Nate Mercaldo, and Dr. Andre Oliveira for statistical evaluation; Dr. Piper Treuting for help with mouse pathology; and Drs. Peter Besmer, David Brooks, Jonathan Fletcher, and Raj Kapur for advice on the article.

Received 3/17/05. Revised 5/11/05. Accepted 5/18/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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