Identification of oncogene and miRNA-regulated programs in myeloid cells

急性髓系白血病(AML)是一组以反复发作的细胞遗传学和/或分子异常为特征的异质性疾病，如表达AML1/MTG8融合蛋白的t(8；21)。然而，在AML中发现的许多改变对白血病发生的功能贡献尚不清楚。MicroRNAs(MiRNAs)以一种复杂而特异的方式调节细胞蛋白质组，但miRNA调控的靶基因及其对造血细胞内信号转导的影响在很大程度上仍不清楚。目前可用的miRNA靶标预测程序容易出错，但将不同的预测算法与途径分析相结合可能会提高miRNA靶标的识别能力。实验上，使用SILAC(细胞培养中氨基酸的稳定同型标记)和LC-MS的蛋白质组分析正在被用来定量miRNA调节的蛋白质表达。我们假设，AML特异的癌蛋白以及miRNAs都调节与疾病进展相关的不同分子程序。在这个项目中，我们的目标是使用一种公正的方法来表征这种程序，以比较AML1/MTG8癌蛋白和miR-100的唯一和重叠的靶点。基于数据(I)在t(8；21)AML细胞中miR-100的表达降低，(Ii)miR-100过表达在t(8；21)Kasumi细胞中的特异性细胞毒性，以及(Iii)在TOR通路中预测多个miR-100靶点，我们建议用SILAC/LC-MS分别研究AML1/MTG8和miR-100过表达和沉默的细胞蛋白质组。利用电穿孔和慢病毒基因转移分别在AML1/MTG8阴性和阳性细胞系中产生瞬时和稳定的和可诱导的AML1/MTG8功能增减表型，并用SILAC/LC-MS分析细胞蛋白质组。同时，miR-100将过度表达，并在AML1/MTG8上存在和不存在的情况下进行SILAC/LC-MS。对调节蛋白的比较，特别是对mTOR途径的成分的比较，将确定AML1/MTG8和miR-100的独特和重叠的靶标。念珠菌靶标将通过基因特异性和剂量等效的RNA干扰(RNAi)进行功能分析。此外，为了改进我们关于原代AML细胞miRNA表达的初步数据，我们将通过miCHIP分析来分析带有和不带有t(8；21)的遗传定义的原代AML细胞中miRNA的表达。这些研究的目的是(I)评估一种新的策略来识别髓系细胞中癌基因和miRNA调控的程序，以及(Ii)更好地表征AML1/MTG8和miR-100在AML t(8；21)中的功能，以便为这一亚型AML寻找新的治疗靶点。

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.