Identification of oncogene and miRNA-regulated programs in myeloid cells

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Acute myeloid leukemia (AML) is a heterogeneous group of diseases characterized by recurrent cytogeneticand/or molecular aberrations such as t(8;21) with expression of the AML1/MTG8 fusion protein. The functionalcontribution to leukemogenesis of many alterations found in AML, however, is not yet understood. MicroRNAs(miRNAs) regulate the cellular proteome in a complex and specific manner, but miRNA regulated target genesand their impact on intracellular signaling in hematopoietic cells are still largely unknown. Currently availablemiRNA target prediction programs are error-prone but the combination of different prediction algorithms linkedto pathway analysis may improve identification of miRNA targets. Experimentally proteome-wide analysesusing SILAC (stable isotype labelling with amino acids in cell culture) and liquid chromatography coupled tomass spectrometry (LC-MS) are being used to quantify miRNA-regulated protein expression. We hypothesizethat both AML-specific oncoproteins as well as miRNAs regulate different molecular programs relevant fordisease progression. In this project we aim to use an unbiased approach to characterize such programsencompassing unique and overlapping targets for the AML1/MTG8 oncoprotein and miR-100. Based on dataon (i) reduction of miR-100 expression in t (8;21) AML cells, (ii) specific cytotoxicity of miR-100over-expression in AML t(8;21) Kasumi cells, and (iii) prediction of multiple miR-100 targets within themTOR-pathway, we propose to study the cellular proteome upon over-expression and silencing ofAML1/MTG8 and miR-100 by SILAC/LC-MS, respectively. Using electroporation and lentiviral gene transfer wewill generate transient as well as stable and inducible AML1/MTG8 gain- and loss-of function phenotypes inAML1/MTG8- negative and positive cell lines, respectively, and analyze the cellular proteome bySILAC/LC-MS. In parallel, miR-100 will be over-expressed, and SILAC/LC-MS will be performed in thepresence and absence on AML1/MTG8. Comparison of regulated proteins with specific focus on componentsof the mTOR pathway will identify unique and overlapping targets of AML1/MTG8 and miR-100. Candidatetargets will functionally be analyzed by gene-specific and dose-equivalent RNA interference (RNAi). Inaddition, to improve our initial data on miRNA expression in primary AML cells, miRNA expression will beanalyzed in genetically defined primary AML cells with and without t(8;21) by miCHIP analysis. These studiesaim (i) to evaluate a new strategy to identify oncogene- and miRNA regulated programs in myeloid cells and (ii)to better characterize the function of AML1/MTG8 and miR-100 in AML t(8;21) in order to identify newtherapeutic targets for this subgroup of AML.