PHENOTYPIC CHARACTERIZATION OF HUMAN NPM1C, DNMT3A MUTATIONS AND THEIR COOPERATION IN ACUTE MYELOID LEUKEMIA USING DROSOPHILA MELANOGASTER MODEL

Abstract

Acute myeloid leukemia (AML) is an aggressive, genetically complex and highly heterogeneous hematological malignancy. Despite increased understanding of the genetic and epigenetic aspects of AML, it still associates with poor prognosis, high relapse rates, and resistance to standard chemotherapy. A better understanding of the molecular pathways involved in single or co-occurring mutations remains essential to offer a better management of AML. Several mutant proteins add intricacy to the prognosis, treatment and management of this disease. Nucleophosmin-1 (NPM1c) is one of the most frequent mutations in AML patients. Normally, NPM1 is a nucleo-shuttling chaperone protein that plays pivotal role in cellular processes. Mutations in NPM1 contribute but are not sufficient to induce AML leukemogenesis. Indeed, NPM1c frequently co-exist with mutant DNA methyltransferase 3A (DNMT3A), an epigenetic modifier involved in de novo DNA methylation, or with FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD), usually worsening the patients’ prognosis. DNMT3A mutations occur in around 20% of AML patients where in the vast majority of cases, mutations are specific to Arg882 (R882H). As part of a bigger project in our laboratory, we employed Drosophila melanogaster to study the phenotypic effect of select single mutations or their co-occurrence. We generated

the targeted human genes: DNMT3A WT or its mutant DNMT3A R882H, NPM1WT or it mutant NPM1c. UAS-Gal4 system was used for eye specific gene expression and transgenic flies expressing phenotypic analysis of the relative eye roughness was evaluated using Scanning Electron Microscopy. Hml-Gal4 system was used for expression of different proteins in the hematopoietic fly compartments, and their phenotypic analysis was performed by hemocyte enumeration. We demonstrated that, upon expression of single mutants in hemocytes of Drosophila, NPM1c induced a slight increase while DNMT3A R882H induced a significant increase in hemocyte count. Similarly, upon the expression of these mutations in the eyes of adult flies, NPM1c showed no change in the eye phenotype whereas DNMT3A R882H caused eye roughness and these changes were specifically at the level of bristles’ organization. Double mutant NPM1c/DNMT3A R882H flies demonstrated an exacerbated and significant phenotype in hemocyte count, as compared to single mutants. Moreover, L3 larvae of the double mutant transgenics exhibited melanotic tumors, reflecting the aggressiveness of the co-occurrence of both mutations. Interestingly, the expression of a double mutation in the eyes of Drosophila led to lethality at pupal stages, which indicates a severity in eye roughness. Our study provides a rapid in vivo assay to identify interactions between different AML mutations, their contribution to disease pathogenesis and therapeutic susceptibilities. It also sets the ground to map the molecular pathways affected by these mutations and break down the pathophysiology of AML to further discover therapeutic susceptibilities and provide insight into personalized medicine approaches.