Assessing the impact of expressing oncogenic BCR-ABL on hematopoiesis and innate immunity in drosophila melanogaster

Abstract

Introduction: CML is caused by a chromosomal translocation resulting in the formation of BCR-ABL fusion gene that encodes a constitutively active BCR-ABL tyrosine kinase which activates multiple signal transduction pathways leading to a myeloproliferative disorder in the bone marrow. Some mutations of the oncogene, especially the substitution of threonine with isoleucine on the 315th residue (T315I mutation) of the ABL kinase, have proven resistant to conventional CML therapy; tyrosine kinase inhibitors (TKI). Transgenic flies harboring wildtype BCR-ABL (P210) and BCR-ABL (T315I) mutants have been generated and tested for their oncogenic potential in a previous study by our group. In this current work, we aimed to investigate the effect of expressing oncogenic BCR-ABL on D. melanogaster hematopoiesis and innate immunity to better understand the impact of this oncogene in the etiology of CML disease in an in vivo model. Methods: BCR-ABLP210 and BCR-ABLT315I were expressed in Drosophila circulating hemocytes which are comparable to mammalian macrophages and bone marrow niche by using the Hemolectin Delta; UAS-GFP (Hml Δ-Gal4; UAS-GFP) driver . The transgene expression was validated by immunofluorescence. Following the overexpression, an analysis of the hematopoietic system was performed by assessing changes in circulating hemocytes and GFP positive cells using hemocytometer and flow cytometry. Moreover, given the cellular role the hemocytes play in the innate immunity in Drosophila melanogaster, the expression of drosomycin, diptericin and Tot A, respective indicators of Toll, IMD and JAK-STAT; the three-major fly humoral immune pathways, were assessed by real time PCR. Results: BCR-ABLP210 and BCR-ABLT315I were expressed in the circulating hemocytes. The number of circulating hemocytes increased significantly in BCR-ABLP210 and BCR-ABLT315I compared to the control. Moreover, the sessile hemocytes patterning was disrupted in both BCR-ABLP210 and BCR-ABLT315I. In addition, melanotic tumors were observed i