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Deliverables
1) The Cys620Arg mutation of RET tyrosine kinase receptor is associated with MEN 2A cancer syndrome and Hirschsprung disease. Mouse model reproducing these pathological phenotypes will be fruitful to study disease appearance, evolution and to develop effective therapeutic tools, for these almost always fatal diseases.
2) We have investigated the effect on TRKA receptor activity of a mutation equivalent to the Met918Thr mutation affecting Ret in MEN2B patients. We have introduced the Met688Thr mutation into both the TRKA receptor and TRK-T3 oncogene cDNAs.
Biochemical and biological studies indicate that, contrariwise to the effect exerted in Ret, theMet688Thr mutation causes inactivation of the TRKA receptor. Since these mutations all occur in a highly conserved TK domain of the studied receptors, the reported contrasting result could have a great importance in a context of drugs aimed at the development of specific inhibitors.
1) The RET proto-oncogene is one of the receptor tyrosine kinases, which are cell-surface molecules that transduce signals for cell growth and differentiation. Mutations in the RET gene are associated with the disorders multiple endocrine neoplasia, type 2A (MEN 2A), multiple endocrine neoplasia, type 2B, Hirschsprung disease (HSCR; aganglionic megacolon) and medullary thyroid carcinoma. The Cys620Arg mutation is associated with both MEN 2A and HSRC exerting a dual effect on RET. It leads to a decrease of the receptor at the cell surface and converts RET into a constitutive activated kinase due to the formation of disulphide-linked homodimers. We have introduced Cys620Arg mutation in cloned RET mouse sequences inserted in a appropriate targeting vector to be introduced in ES (embrional stem) cells. ES cell clones containing Cys620Arg mutation will be selected and used to produce mice carrying the mutation in the heterozygous state.
2) With the final goal to explore the consequences of the constitutive TRKA activation in mouse models, we decided to introduce in TRKA point mutations activating other RTK genes and associated with human cancer. We choose the following mutations: 1) TRKA Met668Thr: is the equivalent of the Ret Met668Thr mutation associated with MEN2B syndrome; 2) TRKA Asp668Val: is the equivalent of the Kit Asp816Val mutation associated with mast cell neoplasms. TRKA and TRK-T3 cDNA carrying the mutations have been produced. Biochemical analysis of mutated proteins expressed into mammalian cells showed that, upon NGF treatment, the TRKA Asp668Val receptor undergoes autophosphorylation, similarly to the wild type; on the contrary, the Met688Thr mutant remains unphosphorylated. The constitutive autophosphorylation of the TRK-T3 oncogene is unaffected by the 668 mutations, whereas is completely abolished by the 688 mutation.
Biological studies in NIH3T3 cells revealed that the transforming activity of wild type receptor in the presence of NGF is retained by the 668 mutant, whereas is completely lost by the 688 mutant. Similarly, the TRK-T3 transforming activity is unaffected by the 668 mutation, whereas it is abrogated by the Met688Thr mutations. Altogether these results indicate that, although occurring in regions conserved among different RTKs, the activated mutations investigated exert different effect on TRKA activity.
Metdominant negative model (Partner 1).
A mutation has been introduced in the kinase domain of the Met receptor which causes the production of a kinase-dead receptor. This mutation has a dominant negative effect, since the kinase-dead product of the mutated allele, by dimerizing with the product of the normal allele, lowers its effective concentration. Thus this mutant mouse has a phenotype in the heterozygous state. The defect is related to the development of the female genito-urinary tract (imperforated vagina). In a mixed genetic background the defect is not fully penetrant. A role for Met in the development of the genito-urinary system had not been previously described. We are re-examining mice with severe and mild loss of function Met alleles (previously generated) to confirm this observation. Similar defects have been described in a rare human syndrome, the Mayer-Rokitansky-Kuster-Hauser syndrome (Griffin et al., Ann Intern Med 85,224-236,1976) in which there is congenital absence of the vagina. We plan to analyse DNA from such patients for Met mutations.
Gain or loss of function mutations in receptor tyrosine kinases /RTKs) generally result in tumorigenesis or in developmental disorders. For Met, it is known that a number of activating mutations in the kinase domain, either inherited or somatically acquired, cause a predisposition to papillary carcinoma of the kidney. On the other end, there is no known congenital defect linked to a partial loss of Met function. A severe loss of Met function (such as that obtained in the knock-out or in a mutant lacking its docking sites) results in a phenotype which is embryonal lethal in homozygosity. These mutants, however, do not show any defect in heterozygosity. We obtained a genetically modified mouse which bears an insertion of four aminoacids right in the middle of the kinase domain, resulting in a kinase-dead receptor. In this case, given the dimerization step in the mechanism of RTKs-mediated signalling, the product of the mutated allele lowers the effective concentration of the product of the wild type allele by forming inactive dimer. This mouse has a phenotype in heterozygosity. Heterozygous female mice occasionally show swelling of the abdomen, due to marked distention of the uterus. The cause for this seems to be accumulation of secretions and pyometra, resulting from an imperforate vagina. This phenotype suggests a new role for Met in the development of the urogenital system. We are very interested in the possible link (suggested by this phenotype) between a partial loss of function of Met and the Mayer-Rokitansky-Kuster-Hauser syndrome (Griffin et al., Ann Intern Med 85,224-236,1976) in which there is congenital absence of the vagina. We will collect DNA sample form these patients to look for Met inactivating mutations.
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