The use of mouse models to help investigate GIST
By Dr. Peter Besmer
Memorial Sloan-Kettering Cancer Center
Dr. Peter Besmer is a member of the LRG Research Team working to understand and oversome GIST treatment resistance. This is the third article in a series to be written by each of the key research team members.
In this newsletter I would like to highlight the importance and usefulness of mice or mouse models in biological and cancer research by using the Kit receptor tyrosine kinase as an example. Receptor tyrosine kinases are molecules which reside in the membrane of cells. They consist of an extracellular domain, a single transmembrane domain and a cytoplasmic domain which includes a tyrosine kinase. Tyrosine kinases are proteins which phosphorylate proteins, or attach phosphate residues, specifically on tyrosine moieties of target proteins. Binding of a ligand to a respective receptor activates the kinase, and this sets in motion distinct signalling events in the receptor expressing cell. Whereas the receptor tyrosine kinase may be expressed in only a few distinct cell populations in the organism/animal, the available signalling machinery available to transmit receptor initiated signals in these different cell types may differ. Thus a receptor initiated signal in different cell types may differ and result in different outcomes.
In our studies of the Kit receptor, a most important observation we had made in collaboration with Alan Bernstein’s group in Toronto was the demonstration that the Kit receptor gene was encoded at the White spotting locus in the mouse (2, 4). Mutations in the White spotting/ Kit gene had been reported quite some time ago due to the effect of the mutation on pigment formation and the consequent lack of pigmentation in these mice – hence the designation white spotting mutation. Previous work had shown that the wild-kit gene had important roles in melanogenesis (the formation of pigment cells), in germ cell development and in hematopoiesis. Mice which lack Kit receptor function lacked coat pigment, they were sterile, and the formation of red blood cells and mast cells were affected. Therefore, the mutant animals died perinatally as a result of the red blood cell deficiency. At the time, the demonstration that Kit was encoded at the white spotting locus was a breakthrough. Subsequent work in the 1990s in great part focused on the role of Kit in hematopoiesis. Today we know that Kit has a role in hematopoietic stem cells, in the progenitors/ precursors of all of the hematopoietic cell lineages, including lymphocytes. In the early 1990s, using a monoclonal antibody which blocks Kit receptor function, Nishikawa’s group in Japan observed that treatment of mice with this monoclonal antibody would cause dysfunctional gastrointestinal motility (9). This then led to the identification and characterization of the interstitial cells of Cajal, which we know today are the cells which may give rise to GIST. Based on phenotypes of Kit mutant mice, the cellular responses which Kit may mediate appear to be quite diverse and include cell proliferation, cell survival (suppression of apoptosis/cell death), cell adhesion, migration, secretory responses and differentiation.
How does the Kit receptor mediate these diverse outcomes? The Kit receptor is known to activate several distinct signalling cascades including phosphatidyl inositol-3 kinase (PI 3-kinase) and Src family kinases (SFK) (See Figure 1).
Both signalling cascades have critical roles in receptor tyrosine kinase signalling and oncogenic transformation. To investigate this question we modified the Kit gene in the mouse genome by substituting critical tyrosine residues in the Kit protein with phenylalanine (1, 5, 8). These substitution mutations in the Kit receptor block either PI 3-kinase or SFK activation (15). The analysis of the phenotypes of the mice carrying these mutations brought to light that PI 3-kinase is critical in male germ cell development, but had no other discernible phenotypes, whereas the SFK mutant mice had defects in hematopoietic cell lineages, but not in germ cell development. These results highlight the critical importance of the cellular environment in which the Kit receptor functions and demonstrates that animal models are critical for elucidating the role and mechanisms of receptor tyrosine kinase signalling in different cell types.
In the 1980s Kit was identified as an oncogene of a feline sarcoma virus (3), but it was only in the 1990s that a role for Kit in human cancer became evident (6, 7, 10). The cancers which are 
associated with oncogenic activation of Kit include most importantly GIST, but oncogenic activation of Kit is also observed in mastocytosis, seminomas, a small subset of AMLs and a small subset of melanomas. In most cancers, mutation or oncogenic activation of the cancer genes occur in somatic cells and thus are only found in one tissue. However, in rare occasions oncogenic mutations are acquired in germ cells and may be transmitted in the germ line (i.e. the oncogenic activation mutation is inherited). Interestingly, some cases of familial GIST have been reported where a Kit gene carrying an oncogenic mutation is inherited (11). We have engineered a mutation found in a familial GIST case into the mouse genome by using homologous recombination approaches. A mouse strain which carries this mutation in the germline has also been obtained (13, 14). Remarkably, these mice recapitulate human familial GIST quite faithfully.
These findings first of all demonstrate that the Kit mutation is the initiating event in the development of familial and presumably non-familial GIST. Secondly, they highlight the unique specificity of the mutant Kit receptor to produce GIST and not other cancers and this implies that the cellular machinery in GIST cells and their microenvironment is quite unique in supporting tumor formation and tumor maintenance. These GIST mice provide a unique opportunity to investigate the mechanism of oncogenic Kit receptor signalling and they provide a superior opportunity to evaluate second generation drugs which might be useful in the treatment of imatinib resistant GIST. In this regard we have been able to identify Kit specific signalling cascades that are inhibited by treatment of these mice with imatinib and begin investigating efficacy of second generation drugs that might be useful to treat imatinib resistant GIST (12). Furthermore, encouraged by our success to recapitulate familial GIST in mice, efforts are now under way to produce a model for imatinib resistant GIST.
References:
1. Agosti, V., S. Corbacioglu, I. Ehlers, C. Waskow, G. Sommer, G. Berrozpe, H. Kissel, C. M. Tucker, K. Manova, M. A. Moore, H. R. Rodewald, and P. Besmer. 2004. Critical role for Kit-mediated Src kinase but not PI 3-kinase signaling in pro T and pro B cell development. J Exp Med 199:867-78.
2. Besmer, P. 1991. The kit ligand encoded at the murine Steel locus: a pleiotropic growth and differentiation factor. Curr Opin Cell Biol 3:939-46.
3. Besmer, P., E. Lader, P. C. George, P. J. Bergold, F. H. Qiu, E. E. Zuckerman, and W. D. Hardy. 1986. A new acute transforming feline retrovirus with fms homology specifies a C-terminally truncated version of the c-fms protein that is different from SM-feline sarcoma virus v-fms protein. J Virol 60:194-203.
4. Besmer, P., J. E. Murphy, P. C. George, F. H. Qiu, P. J. Bergold, L. Lederman, H. W. Snyder, Jr., D. Brodeur, E. E. Zuckerman, and W. D. Hardy. 1986. A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family. Nature 320:415-421.
5. Blume-Jensen, P., G. Jiang, R. Hyman, K. F. Lee, S. O'Gorman, and T. Hunter. 2000. Kit/stem cell factor receptorinduced activation of phosphatidylinositol 3'-kinase is essential for male fertility. Nat Genet 24:157-62.
6. Hirota, S., K. Isozaki, Y. Moriyama, K. Hashimoto, T. Nishida, S. Ishiguro, K. Kawano, M. Hanada, A. Kurata, M. Takeda, G. Muhammad Tunio, Y. Matsuzawa, Y. Kanakura, Y. Shinomura, and Y. Kitamura. 1998. Gain-offunction mutations of c-kit in human gastrointestinal stromal tumors. Science 279:577-80.
7. Kemmer, K., C. L. Corless, J. A. Fletcher, L. McGreevey, A. Haley, D. Griffith, O. W. Cummings, C. Wait, A. Town, and M. C. Heinrich. 2004. KIT mutations are common in testicular seminomas. Am J Pathol 164:305-13.
8. Kissel, H., I. Timokhina, M. P. Hardy, G. Rothschild, Y. Tajima, V. Soares, M. Angeles, S. R. Whitlow, K. Manova, and P. Besmer. 2000. Point mutation in Kit receptor tyrosine kinase reveals essential roles for Kit signaling in spermatogenesis and oogenesis without affecting other Kit responses. Embo J 19:1312-1326.
9. Maeda, H., A. Yamagata, S. Nishikawa, K. Yoshinaga, S. Kobayashi, and K. Nishi. 1992. Requirement of c-kit for development of intestinal pacemaker system. Development 116:369-75.
10. Nagata, H., A. S. Worobec, C. K. Oh, B. A. Chowdhury, S. Tannenbaum, Y. Suzuki, and D. D. Metcalfe. 1995. Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Natl Acad Sci U S A 92:10560-4.
11. Nishida, T., S. Hirota, M. Taniguchi, K. Hashimoto, K. Isozaki, H. Nakamura, Y. Kanakura, T. Tanaka, A. Takabayashi, H. Matsuda, and Y. Kitamura. 1998. Familial gastrointestinal stromal tumours with germline mutation of the KIT gene [letter]. Nat Genet 19:323-4.
12. Rossi, F., I. Ehlers, V. Agosti, N. D. Socci, A. Viale, G. Sommer, Y. Yozgat, K. Manova, C. R. Antonescu, and P. Besmer. 2006. Oncogenic Kit signaling and therapeutic intervention in a mouse model of gastrointestinal stromal tumor. Proc Natl Acad Sci U S A 103:12843-8.
13. Rubin, B. P., C. R. Antonescu, J. P. Scott-Browne, M. L. Comstock, Y. Gu, M. R. Tanas, C. B. Ware, and J. Woodell. 2005. A knock-in mouse model of gastrointestinal stromal tumor harboring kit K641E. Cancer Res 65:6631-9.
14. Sommer, G., V. Agosti, I. Ehlers, F. Rossi, S. Corbacioglu, J. Farkas, M. Moore, K. Manova, C. R. Antonescu, and P. Besmer. 2003. Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase. Proc Natl Acad Sci U S A 100:6706-11.
15. Timokhina, I., H. Kissel, G. Stella, and P. Besmer. 1998. Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation. Embo J 17:6250-6262.
