The overall objective of this proposal is twofold, 1) to continue our investigations of oncogenic Kit receptor signaling in vivo with emphasis on hematopoiesis and 2) to develop mouse models for imatinib resistant GIST. The Kit receptor encoded at the murine W locus functions in hematopoiesis, gametogenesis, melanogenesis and gut motility. Normal Kit receptor mediated functions include cell proliferation, cell survival, cell adhesion, cell migration, secretory responses and differentiation. In human neoplasia oncogenic activation of Kit has roles in gastrointestinal stromal tumors (GIST), mastocytosis/mast cell leukemia, acute myelogenous leukemia, a subset of melanomas and a subset of germ cell tumors. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules and signaling cascades. How do receptor tyrosine kinases such as Kit mediate distinct cellular responses in different cell types during embryonic development and in the postnatal animal? And what are the requirements for oncogenic transformation in different cell types to produce cancer. We have produced mice containing knock-in point mutations in the Kit receptor gene in mice which block Kit mediated PI 3-kinase activation and signaling or Src family kinase, SFK, activation and signaling. These mice have distinctly different phenotypes in gametogenesis and hematopoiesis. We have also investigated the role of PI 3-kinase and Src family kinase signaling in Kit mediated cell proliferation, suppression of apoptosis, cell adhesion, chemotaxis and secretory responses in vitro in bone marrow derived mast cells (BMMC). Whereas Kit mediated PI 3-kinase signaling is critical for cell proliferation, cell survival, cell adhesion, chemotaxis and secretory responses, Kit mediated SFK signaling has mostly a negative regulatory role. Furthermore, gene expression profiling indicates that blocking SFK signaling reduces KitL induced expression of TH2-cytokine genes in BMMC. To investigate the role of oncogenic Kit activation Kit in tumorigenesis we have produced mice carrying a juxtamembrane domain knock-in Kit mutation and reproduced human familial GIST syndrome (Sommer et al., 2003). In part our proposed studies will attempt to investigate the role of PI 3-kinase, SFK and Ras signaling in Kit-mediated tumorigenesis. Furthermore we will produce and characterize mice carrying oncogenic imatinib resistant Kit alleles.
The Kit receptor has roles in pigment formation, hematopoiesis, germ cell development and pacemaker cells of the gastrointestinal tract in interstitial cells of Cajal. 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. Furthermore, Kit has roles in human cancer. 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. How does the Kit receptor mediate these diverse outcomes? The Kit receptor is known to activate several distinct signaling cascades including phosphatidyl inositol-3 kinase (PI 3-kinase) and Src family kinases (SFK). Both signaling cascades have critical roles in receptor tyrosine kinase signaling and oncogenic transformation. To investigate this question we have modified the Kit gene in the mouse genome by substituting critical tyrosine residues in the Kit protein with phenylalanine. These substitution mutations in the Kit receptor block either PI 3-kinase or SFK activation. 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 or cellular context in which the Kit receptor functions and that animal models are critical for elucidating the role and mechanisms of receptor tyrosine kinase signaling in different cell types. In most cancers oncogenic Kit receptor mutations occur in somatic cells. However, in rare occasions oncogenic Kit mutations are acquired in germ cells and are transmitted in the germ line. We have engineered a mutation found in a familial GIST case into the mouse genome by using homologous recombination approaches and a mouse strain which carries this mutation in the germline has been obtained. These mice recapitulate human familial GIST quite faithfully. These findings demonstrated that the Kit mutation is the initiating event in the development of familial and presumably non-familial GIST. Second 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 signaling and they provide an opportunity to evaluate second generation drugs which might be useful in the treatment of imatinib resistant GIST. As part of this proposal first we are investigating the role of the oncogenic Kit-V558 deletion mutation in hematopoiesis and in hematologic malignancies. Second we will investigate the mechanism of oncogenic signaling deriving Kit mutant mice containing an oncogenic Kit mutation but which fail to activate SFK or PI 3-kinase signaling. Today patients with GIST are successfully treated with the kinase inhibitor imatinib. However most patients eventually become resistant to drug treatment as a result of the acquisition of a secondary Kit mutation. Encouraged by our success to recapitulate familial GIST in mice, as part of this proposal we will to produce mouse models for imatinib resistant GIST.
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