PHENOTYPIC CHARACTERIZATION OF HUMAN NPM-1C, IDH1, IDH2, AND P53 MUTATIONS AND THEIR COOPERATION IN ACUTE MYELOID LEUKEMIA USING DROSOPHILA MELANOGASTER MODEL

Abstract

Acute myeloid leukemia (AML) is a genetically complex and highly heterogeneous hematological malignancy. Despite the increasingly garnered molecular knowledge, AML still associates with poor prognosis, high relapse rates, and resistance to standard chemotherapy. Unraveling the molecular pathways of single or co-occurring mutations is indispensable to improve AML management. under a broader research by our team investigating relevant mutations in AML, we focused on select mutated genes. Mutations in isocitrate dehydrogenase 1 or 2 (IDH 1/2) encoding for two known epigenetic regulators and key enzymes of the Krebs cycle, occur in AML. These mutations result in an aberrant enzymatic function, DNA hypermethylation, cell proliferation and abnormal differentiation. Nucleophosmin-1 is a phosphoprotein playing a pivotal role in several cellular processes and its gene mutations (NPM-1c) classify among the most frequent mutations in AML patients. NPM-1c contributes but is not sufficient to induce AML. Its occurrence with other mutations is key in AML pathophysiology. Finally, P53, a well-known tumor suppressor, is mutated in 5 to 10% of AML patients. We utilized Drosophila melanogaster model to study the phenotypic effect of single mutations IDH1 R132H, IDH2 R172K, NPM1c, and P53 R248Q and IDH1 R132H/ NPM1c double mutation. Transgenic flies were generated by injecting pUAST vectors carrying the targeted wild-type human genes or their relevant mutations in AML. Hemolectin-Gal4 system was used to express different proteins in the hematopoietic fly compartments, and their phenotypic analysis was performed by hemocyte enumeration. UAS-Gal4 system was used to specifically drive expression to the eyes of adult flies. Phenotypic analysis was evaluated by Scanning Electron Microscopy. We demonstrated that expression of IDH2 R172K or IDH1 R132H mutations increase the number of circulating hemocytes and that IDH1 R132H induces moderate roughness in the eyes of adult transgenic flies. NPM1c mutation increases hemocyte count but does not induce any eye roughness in adults. Interestingly, IDH1 R132H/ NPM1c double mutation significantly increased circulating hemocytes counts as compared to single mutations. Finally, and surprisingly, P53 R248Q mutation decreases hemocyte count. Our study further validates the use of Drosophila to study mutations of AML and sets a solid ground to map the molecular pathways by which these mutations exert their biological phenotypes and to discover therapeutic susceptibilities providing insights towards personalized medicine approaches.