Identification of Genes Sustaining Bcr-Abl Oncogenic Signalling and CML Progression through a Genetic Tool Based on Human Bcr-Abl Transgenic Drosophila Melanogaster.

Although the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukaemia (CML) is well established, the mechanisms responsible for CML progression are largely unknown. The aims of the study were

• to perform a genetic screening to identify new genes and pathways leading to CML progression and imatinib resistance and

• to provide a powerful tool allowing a wide screening of drug libraries.

We developed a genetic model based on transgenic human p210 Bcr-Abl Drosophila melanogaster (Dm). We generated two different fly lines expressing either h-p210 wt or carrying the T315I mutation, in a tissue specific manner such as fly eyes or lymph gland, which represents the Dm hematopoietic system. Bcr-Abl expression results into a glazed phenotype of the eyes correlated with the amount of p210 protein. A wide modifier screening was performed using commercially available stocks of Dm carrying small and well characterized chromosome deletions. The resulting progeny was first screened using eye phenotype as read-out system. A first group of flies displayed a more aggressive phenotype since they lack one or more genes encoding for Bcr-Abl negative regulators while a second group displayed a mild phenotype most likely due to the absence of a gene encoding for a gene involved in the oncogenic signalling. Each deletion, responsible for any phenotype change in the progeny, was further analyzed by crossing Bcr-Abl flies with flies carrying the single deletion of each gene included in the identified region. Among the genes identified, PI3K loss of function results into a phenotype improvement thus supporting the tool effectiveness. As control, the cross between Bcr-Abl flies and the constitutively active form of PI3K results not only into a worse phenotype but also into an increased size of the eye, corresponding to an abnormal proliferation process. With this tool we have at now identified a list of genes responsible for a phenotype change including Fax, Dab and Pros which have been more extensively studied in human primary cells collected from patients at diagnosis enrolled in TOPS studies (Cortes, EHA, 2008) and during disease progression, so confirming their involvement in human disease. We found that these genes are downregulated during accelerated phases and blast crisis. Transfection of CML cells with Fax, Dab and Pros reduced proliferation and/or induced apoptosis. By contrast, loss of function of ENA, the CRKL hortologous in Dm did not induce any significant change of the phenotype. In addition, a drug screening was performed by feeding flies with drugs. We set up a rapid method for drug testing based on wt and T315I/Bcr-Abl phenotypes rescue induced by several TKs inhibitors or by combinations. In conclusion, Dm is a rapid genetic tool which allow the selection of a number of genes involved in CML progression and IM resistance. In addition, it allows to identify drugs or combination of drugs active on Bcr-Abl or T315I mutant form. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology