Proteomic analysis of AML1/RUNX1 mutant complexes

骨髓增生异常综合征（MDS）是一种以造血功能低下和不足为特征的造血干细胞克隆性疾病。MDS的一部分患者是在既往化疗或其他恶性肿瘤放疗后出现的。由于MDS是一种异质性疾病，在骨髓增生异常过程中起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AML1/RUNX1基因的体细胞突变，该基因是决定性造血的关键调节因子，也是MDS患者急性髓性白血病（AML）易位的最常见靶点。我们在110例难治性贫血伴过多原细胞（RAEB）、转化中的RAEB （RAEBt）和MDS后AML（将这些类别定义为MDS/AML）患者中的26例（23.6%）中检测到AML1点突变。在22例与辐射相关（包括14例原子弹幸存者）和/或治疗相关的MDS/AML患者中，11例（50%）患者AML1突变主要发生在n端re…More区域。相比之下，88例散发性MDS/AML患者中有15例（17%）在n端和c端区域均显示AML1突变。AML1突变的MDS/AML患者预后明显差于无AML1突变的患者。大多数AML1突变体失去了反式激活电位，无论其DNA结合电位如何。这些数据表明，AML1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。为了阐明AML1功能障碍的机制，我们建立了稳定表达flag标记的野生型或突变型AML1蛋白的细胞系。用抗flag抗体免疫沉淀纯化AML1蛋白转录因子复合物，然后用MALDI-TOF/TOF分析仪进行分析。我们在与野生型AML1的复合体中发现了CBFβ、ets-1、p300、C/EBPα和GATA-1，以及之前使用不同方法的研究。然而，在突变的aml1复合体中发现了其他蛋白质。我们一直在分析AML1与这些蛋白的直接结合能力。少

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