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Proteomic analysis of AML1/RUNX1 mutant complexes

AML1/RUNX1 突变体复合物的蛋白质组学分析

基本信息

批准号：
15591008
负责人：
HARADA Hironori
金额：
$ 2.24万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591008/
关键词：
AML1 RUNX1 myelodysplastic syndrome (MDS)Point mutations

项目摘要

Myelodysplastic syndrome(MDS) is a clonal disorder of hematopoietic stem cells characterized by ineffective and inadequate hematopoiesis. MDS in a subset of patients arise after previous chemotherapy or radiation exposure for other malignancies. As MDS is a heterogeneous disorder, specific gene abnormalities playing a role in the myelodysplastic process have been difficult to identify. In this study, we analyzed the somatic mutations in the AML1/RUNX1 gene, which is a critical regulator of definitive hematopoiesis and the most frequent targets for translocation of acute myeloid leukemia (AML), in patients with MDS. We detected AML1 point mutations in 26 of 110(23.6%) patients with refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt) and AML following MDS (defined these categories as MDS/AML). Among 22 patients with radiation-related (including 14 atomic bomb survivors) and/or therapy-related MDS/AML, 11(50%) patients had the AML1 mutations mostly in N-terminal re … More gion. In contrast, 15 of 88(17%) patients with sporadic MDS/AML showed the AML1 mutations equally in both N-terminal and C-terminal region. The MDS/AML patients with AML1 mutations had a significantly worse prognosis than those without AML1 mutations. Most of AML1 mutants lost trans-activation potential, regardless of their DNA binding potential. These data suggested that AML1 point mutation is one of the major driving forces of MDS/AML, and these mutations may represent a distinct clinicopathologic-genetic entity.To clarify mechanisms of the AML1 dysfunctions, we established cell lines stably expressing FLAG-tagged wild-type or mutant AML1 proteins. A complex of transcriptional factors including these AML1 proteins were purified by immunoprecipitation with an anti-FLAG antibody, and then were analyzed by MALDI-TOF/TOF analyzer. We identified CBFβ,ets-1,p300,C/EBPα and GATA-1 in the complex with wild-type AML1 as well as previous studies using different methods. However, other proteins were identified in the complex with mutated-AML1. We have been analyzing the direct binding ability of AML1 to these proteins. Less

骨髓增生异常综合征（MDS）是一种造血干细胞克隆性疾病，其特征是无效和不充分的造血。MDS在一个子集的患者出现后，以前的化疗或放射暴露的其他恶性肿瘤。由于MDS是一种异质性疾病，在骨髓增生异常过程中发挥作用的特定基因异常一直难以确定。在这项研究中，我们分析了AML 1/RUNX1基因的体细胞突变，这是一个决定性造血的关键调节因子，也是MDS患者中急性髓性白血病（AML）易位的最常见靶点。我们在110例难治性贫血伴原始细胞过多（RAEB）、RAEB转化（RAEBt）和MDS后AML（定义为MDS/AML）患者中检测到26例（23.6%）AML 1点突变。在22例放射相关和/或治疗相关的MDS/AML患者中，11例（50%）患者存在AML 1突变，主要发生在N末端，11例（50%）患者存在AML 1突变。 ...更多信息吉翁。而88例散发性MDS/AML患者中有15例（17%）在N端和C端均出现AML 1突变。AML 1基因突变的MDS/AML患者的预后明显差于AML 1基因突变的MDS/AML患者。大多数AML 1突变体失去了反式激活潜力，无论其DNA结合潜力。这些结果表明，AML 1点突变是MDS/AML的主要驱动力之一，这些突变可能代表了一种独特的临床病理-遗传实体。用抗FLAG抗体免疫沉淀法纯化包含这些AML 1蛋白的转录因子复合物，然后用MALDI-TOF/TOF分析仪分析。我们在与野生型AML 1的复合物中鉴定了CBF β、ets-1、p300、C/EBP α和加塔-1，以及使用不同方法的先前研究。然而，在与突变型AML 1的复合物中鉴定出其他蛋白质。我们一直在分析AML1与这些蛋白质的直接结合能力。少