New pharmacological targets in Fanconi Anemia

Fanconi Anemia (FA) is a cancer predisposition syndrome with cells sensitivity to interstrand crosslinking (ICL) agents such as cisplatin (CDDP). Genes in the FA pathway are evolutionary conserved, allowing genetic and molecular studies in the model system C.elegans. The FA pathway is involved in the choice between the high fidelity repair pathway, Homologous Repair (HR), and the error prone Non-Homologous End Joining (NHEJ). In C. elegans FCD-2 (the orthologue of the human FACD2) suppresses the use of NHEJ pathway and prevents DNA repair defects (Adamo et al. 2010). In order to confirm this data and determine the effect of the NHEJ block, ICL sensitivity assays were performed in FA patients' cell lines harbouring mutations in FANCC (ADLAT) and treated with siRNA against different components of NHEJ pathway. Preliminary data suggest that the inhibition of KU80 decreases the ICL sensitivity partially restoring the wild type phenotype. clt-2 (cross-linking-tolerance) is a spontaneous recessive mutation we identified in a fcd-2 background for its ability to suppress the CDDP hyper-sensitivity of fcd-2. The average number of meiotic RAD-51 foci and apoptotic nuclei in untreated worms decreases in the clt-2;fcd-2 compared to fcd-2 excluding the possibility that clt-2 is implicated in CDDP uptake. clt-2 mutation significantly reduces the percentage of developmental defects occurring in the fcd-2 population. In our previous studies the same phenomenon was observed in absence of functional NHEJ in lig-4;fcd-2. However, no mutations in known genes of the NHEJ pathways were observed in clt-2. Furthermore, unlike in fcd-2;lig-4 and lig-4 mutants, where the NHEJ is defective, in the clt-2;fcd-2 and fcd-2 mutants the NHEJ works at levels similar to the wild-type in late embryogenesis. Unlike in fcd-2 strains, however, no up-level of NHEJ was observed during meiosis in the clt-2;fcd-2. The mutation has genetically been mapped on chromosome II and genomic sequencing has restricted the region to 800 Mb by linkage mapping. To identify novel players in ICL repair pathway choice we have undertaken a parallel biochemical and proteomic approach. We have obtained an antibody directed against the C-terminal domain of the C. elegans FCD-2 protein. The antibody recognizes both the endogenous and the heterologously expressed protein in its native as well as denatured forms and thus is suitable to the identification of FCD-2 interacting partners. To study the expression and regulation of these proteins we have set up proteomic analysis methods in C. elegans for differentially expressed proteins in response to ICL damage. Recombinant forms of the full-length FCD-2 and its interacting partner FANC-I proteins have been expressed in E. coli, along with a collection of deletion mutants encoding different domains of both proteins. The study of the biochemical activities of these proteins and their interactions by in vitro qualitative and quantitative assays is underway.