Leukemic transformation by the AML1/MDS1/EVI1 Protein

描述（由申请人提供）：我们治愈白血病的希望取决于我们对白血病发生的分子机制的理解。这一点可以从白血病不同亚群的非随机染色体易位研究中获得。t（3;21）发生在治疗相关性AML、CML的原细胞危象期和新生AML的情况下，并且与特别差的预后相关。这种易位导致产生190 kDa的AML1/MDS1/ evi （AME）融合蛋白。该蛋白已被证明在将转导的骨髓细胞移植到辐照受体后诱导小鼠白血病，并通过一系列重复试验评估小鼠骨髓细胞的长生不老性。这种蛋白质转化细胞的机制尚不清楚。我们提出解决这一机制的三个具体目标。第一个目标是使用体外复制试验来评估缺乏关键功能域的AME突变体的转化能力。已知AME在蛋白质的AML1， MDS1和EVIl部分包含许多结构域。我们将测试这些突变体在体外转化小鼠骨髓细胞的能力，使用连续复制实验，以确定哪些功能对转化很重要。第二个目标是确定转化的造血细胞中含有AME的蛋白质复合物的成分。我们已经创建了一种c端标记的AME版本，能够通过体外复制试验进行转化。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物。我们将从表达转化突变形式AME的细胞中纯化复合物。这将允许我们识别对转换至关重要的交互。使用质分光光度法，我们将确定这些配合物的成分。第三个目标是利用短发夹抑制rna下调AME转化造血细胞中的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种可诱导的RNA系统，以实现AME的快速抑制。这与时间过程微阵列分析相结合，将使我们能够以一种时间方式确定AME丢失后基因表达的变化。

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.