Development of a Genetic Tool Based on BCR-ABL Transgenic Drosophila Melanogaster for Identification of Genes Involved in CML Progression and Drug Testing.

Although the role of Bcr-Abl in the pathogenesis of chronic myeloid leukaemia is well established, the molecular mechanisms by which it triggers cellular transformation remains still partially unknown. In addition, the mechanisms responsible for CML progression and the molecules interacting with Bcr-Abl are largely unknown. and many of. In this study, we have developed a novel approach for genetic analysis investigations to identify a number of candidate genes and pathways involved in disease progression and imatinib resistance by using a model of the Drosophila melanogaster. We generated two different stable transgenic fly lines expressing both human p210Bcr-Abl forms (either w.t. or the mutated form T315I) in a tissue specific manner. We have observed that the activation of BCR-ABL led to a particular phenotype in the fly eyes. Transgenic flies will be phenotipically and genotipically characterized carefully by analyzing the eye development. We conducted an extensive genetic screening using both Drosphila transgenic lines overexpressing human Bcr-Abl forms (Bcr-Abl/w.t. and Bcr-Abl/T315I) by making use of P-elements. This technique offers the possibility to randomly insert P-elements into the genome, thus disrupting genomic loci either in correspondence of coding sequences or regulatory elements and altering the gene function. The heterogeneous mutagenized fly population was carefully screened on the basis of the flies phenotype. The first group which we have selected was represented by flies displaying a phenotype even more aggressive than the parental transgenic fly. This kind of flies harbour mutated genes encoding for proteins which enhance the activity of Bcr-Abl, thus being most likely involved in disease progression; A second group was represented by flies displaying a wild-type phenotype. In the latter case the phenotype reversion most likely due to a mutation occurred at a level of a gene encoding for a protein functioning as Bcr-Abl negative regulator. All the data obtained with the use of fly model were confirmed in both cell lines and in primary cells via the overexpression and/or silencing of the genes identified with the Drosophila genetic-screening. Finally we have established and validated a novel tool for drug testing based on the examination of the eye phenotype induced by Bcr-Abl, which can be reverted by the drugs producing a complete block of the kinase activity. With this method we will be able to screen drug libraries to identify molecules able to silence Bcr-AblT315I tyrosine kinase activity. That can be easily accomplished by feeding flies with food previously mixed with the different drug molecules. Molecules showing a good inhibitory activity can be quickly identified because their /ability to revert the abnormal eye phenotype displayed by the transgenic flies. In conclusion, by using a model of the Drosophila melanogaster we have developed a system to identify novel genetic pathways which regulate the development and the progression of CML and we have shown that this novel system can be also used to evaluate the drugs affecting Bcr-Abl regulatory pathway. Moreover, this system does not require a priori knowledge of the function of the genes involved in the disease.