Function and targeting of a stable transcription factor complex in leukemia

描述(申请人提供)：转录因子在许多类型的癌症中发挥关键作用，但长期以来，由于缺乏可被大多数当前药物典型的小分子类型靶向的特定活性部位，因此被认为是“无法用药的”。然而，由于不同的相互作用模式可能赋予多肽高度的选择性，使用短肽最近已成为靶向通过特定蛋白质复合体发挥功能的转录因子的一种有前途的策略。在这项建议中，我们试图使用这一新策略来靶向急性髓系白血病最常见的白血病融合蛋白/转录因子AML1-ETO。我们发现，在白血病细胞中，AML1-ETO存在于包含多种转录因子和辅因子的稳定蛋白质复合体中。在这个复合体中，二聚化的AML1-ETO直接与一系列传统的转录激活因子E蛋白相互作用，E蛋白与造血谱系发育事件有关。我们还发现，AML1-ETO二聚化结构域(NHR2)是白血病发生的关键因素，它利用二聚化的α-螺旋的独特表面来调节AML1-ETO与E蛋白中保守基序的相互作用。这种特殊的相互作用模式理想地允许设计抑制剂来专门干扰相互作用并操纵AML1-ETO的活性，从而为白血病治疗提供一个潜在的靶点。在这方面，为了进一步支持这种NHR2-E蛋白相互作用作为治疗靶点，已经证明，一个特定的突变破坏了NHR2-E蛋白相互作用，但不是NHR2二聚体，削弱了AML1-ETO促进人类造血干细胞自我更新的能力。基于对AML1-ETO转录因子/辅因子(AETFC)复合体的生化和功能研究，我们计划(I)通过详细的机制研究进一步鉴定和表征AETFC复合体；(Ii)通过全基因组芯片分析确定AML1-ETO的直接靶基因；(Iii)通过基于细胞和无细胞的体外转录系统，阐明AML1-ETO和其他组分协同(去)调控转录的详细机制；(Iv)研究(和验证)白血病细胞和小鼠模型中单个成分的生物学功能及其相互作用；以及(V)设计特定的模拟肽抑制剂来操纵AML1-ETO在转录和白血病发生中的作用。 与公共卫生相关：许多染色体易位导致白血病融合蛋白改变正常转录程序。这项研究将对急性髓系白血病中最常见的致白血病融合蛋白(AML1-ETO)可能的不同功能和作用机制提供更深入的了解。这些信息将被用来开发一种针对这种白血病融合蛋白的模拟肽抑制剂，并为相关白血病提供一种新的治疗方法。

DESCRIPTION (provided by applicant): Transcription factors play critical roles in many types of cancer but, for a long time, were considered "undruggable" because of the lack of specific active sites that can be targeted by the types of small molecules that typify most current drugs. However, the use of short peptides has recently emerged as a promising strategy to target transcription factors that function through specific protein complexes, since distinct interaction patterns may confer a high degree of selectivity on the peptide. In this proposal, we seek to use this new strategy to target the leukemogenic fusion protein/transcription factor, AML1-ETO, that is most frequently involved in acute myeloid leukemia. We have found that, in leukemic cells, AML1- ETO resides in a stable protein complex containing multiple transcription factors and cofactors. Within this complex, the dimerized AML1-ETO directly interacts with a family of conventional transcriptional activators, E proteins, that are implicated in hematopoietic lineage developmental events. We also have found that the AML1-ETO dimerization domain (NHR2), which previously was shown to be critical for leukemogenesis, utilizes a distinct surface of the dimerized alpha-helixes to mediate the AML1-ETO interaction with a conserved motif in E proteins. This particular interaction pattern ideally allows the design of inhibitors to specifically disrupt the interaction and to manipulate the activities of AML1-ETO, thus providing a potential target for leukemia treatment. In this regard, and in further support of this NHR2-E protein interaction as a therapeutic target, a specific mutation that disrupts the NHR2-E protein interaction, but not NHR2 dimerization, has been shown to impair the capacity of AML1-ETO to enhance human hematopoietic stem cell self-renewal. Based on these biochemical and functional studies of the AML1-ETO-containing transcription factor/cofactor (AETFC) complex(es), we plan (i) to further identify and characterize the AETFC complex(es) by detailed mechanistic studies; (ii) to identify direct AML1- ETO target genes by genome-wide ChIP analyses; (iii) to clarify, through cell-based and cell-free in vitro transcription systems, the detailed mechanisms by which AML1-ETO and other components cooperate to (de)regulate transcription; (iv) to study (and validate) the biological functions of individual components and their interactions in leukemic cellular and mouse models; and (v) to design specific peptidomimetic inhibitors to manipulate the action of AML1-ETO in transcription and leukemogenesis. PUBLIC HEALTH RELEVANCE: A number of chromosomal translocations result in leukemogenic fusion proteins that alter normal transcription programs. This study will provide a deeper understanding of the likely diverse functions and mechanisms of action of the leukemogenic fusion protein (AML1-ETO) that is most frequently involved in acute myeloid leukemia. This information, in turn, will be used to develop a peptidomimetic inhibitor that targets this leukemogenic fusion protein and offers a new therapeutic approach for related leukemias.