Leukemic transformation by the AML1/MDS1/EVI1 Protein

AML1/MDS1/EVI1 蛋白引起的白血病转化

• 批准号: 6862010
• 负责人: Archibald S. Perkins
• 金额: $ 30.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6862010
• 关键词: RNA interference; acute myelogenous leukemia; affinity chromatography; bioassay; carcinogenesis; chimeric proteins; chromosome translocation; hematopoietic stem cells; laboratory mouse; leukemia; mass spectrometry; microarray technology; neoplasm /cancer genetics; neoplastic transformation; oncoproteins; protein purification; protein structure function; tissue /cell culture

DESCRIPTION (provided by applicant): Our hope of curing leukemia rests on our understanding of the molecular mechanisms involved in leukemogenesis. Insight into this can be garnered from the study of nonrandom chromosomal translocations that occur in different subsets of leukemia. The t(3;21) occurs in the setting of therapy-related AML, the blast crisis phase of CML, and de novo AML, and is associated with a particularly poor prognosis. This translocation results in the production of the 190 kDa AML1/MDS1/EVII (AME) fusion protein. This protein has been shown to induce leukemia in mice following transplantation of transduced bone marrow cells into irradiated recipients, and to immortalize murine bone marrow cells as assessed by a serial replating assay. The mechanism by which this protein transforms cells is not known. We propose three specific aims to address this mechanism. The first goal is to use in vitro replating assay to assess the transforming capability of AME mutants that lack key functional domains. AME is known to contain a number of domains within the AML1, MDS1 and EVIl portions of the protein. We will test the ability of these mutants to transform mouse bone marrow cells in vitro, using the serial replating assay, to determine which of these functions is important for transformation. The second goal is to determine the components of protein complexes that contain AME from transformed hematopoietic cells. We have created a C-terminally tagged version of AME that is capable of transforming as assessed by the in vitro replating assay. This tag is tandem affinity purification (TAP) tag allows for rapid purification of protein complexes from crude cell extracts. In we will purify complexes from cells expressing transforming mutant forms of AME. This will allow us to identify interactions that are essential for transformation. Using mass spectrophotometric techniques, we will determine the components of these complexes. The third goal is to use short hairpin inhibitory RNAs to downregulate AME in AME-transformed hematopoietic cells, and determine the changes in gene expression by microarray analysis. We will also develop an inducible RNA system in AME-transformed cells, to allow rapid suppression of AME. This, in conjunction with time course microarray analysis, will allow us to determine, in a temporal manner, the changes in gene expression that ensue from loss of AME.

描述(申请人提供)：我们治愈白血病的希望寄托在我们对白血病发生的分子机制的理解上。通过对白血病不同亚群中发生的非随机染色体易位的研究，可以获得对这一点的洞察。T(3；21)发生在治疗相关AML、CML急变期和初发AML的背景下，与预后特别差有关。这种易位导致产生190 kDa的AML1/MDS1/EVII(AME)融合蛋白。这种蛋白已被证明在将转导的骨髓细胞移植到受照射的受者体内后诱导小鼠发生白血病，并通过一系列重复试验评估使小鼠骨髓细胞永生化。这种蛋白质转化细胞的机制尚不清楚。我们提出了三个具体目标来解决这一机制。第一个目标是使用体外复制试验来评估缺乏关键功能结构域的AME突变体的转化能力。已知AME在AML1、MDS1和蛋白质的邪恶部分中包含许多结构域。我们将使用连续重复实验测试这些突变体在体外转化小鼠骨髓细胞的能力，以确定这些功能中的哪些对转化是重要的。第二个目标是从转化的造血细胞中确定含有AME的蛋白质复合体的成分。我们已经创造了一个C末端标记的AME版本，它能够通过体外复制实验进行转化。该标签是串联亲和纯化(TAP)标签，允许从粗细胞提取物中快速纯化蛋白质复合体。我们将从表达AME的转化突变形式的细胞中纯化复合体。这将使我们能够确定对转型至关重要的交互作用。使用质量分光光度技术，我们将确定这些络合物的组成。第三个目标是利用短发夹状抑制RNA下调AME转化的造血细胞中的AME，并通过微阵列分析确定基因表达的变化。我们还将在AME转化的细胞中开发一个可诱导的RNA系统，以允许快速抑制AME。这与时间进程微阵列分析相结合，将使我们能够以时间的方式确定AME丢失后基因表达的变化。