Function and targeting of a stable transcription factor complex in leukemia

项目摘要 发育是由调控基因的转录因子的适时表达和功能控制的 表达并决定细胞命运和功能。白血病通常由染色体易位引起， 导致产生新的转录因子，由两个独立的基因的异常融合产生 产品.在急性骨髓性白血病（AML）中，最常见的这种融合是t（8;21）衍生的AML 1-AML融合。 ETO（AE），它通过选择其他正常细胞转录因子来驱动白血病，这些转录因子对 造血干细胞自我更新和增殖，从而使细胞陷入增殖的不成熟状态， 白血病的基础已经证明很难抑制转录因子的功能， 我们需要寻找更易处理的药物靶点的重要AE辅因子。我们的目标是将联合收割机 解释AML基因调控机制的实验方法，包括鉴定 不同监管层级的新参与者将揭示治疗的新机会。 在最近的研究中，我们鉴定了AE相关的转录因子/辅因子复合物（AETFC）， 确定了AE和E蛋白之间的一种新的相互作用表面，这对AETFC功能至关重要， 白血病发生最重要的是，我们在小鼠模型中发现，这种相互作用的破坏严重影响了小鼠的免疫功能。 降低AE致癌性。此外，我们还确定了JMJD 1C作为一种新的AETFC的共活化剂。JMJD 1C是 特别感兴趣的是，它是具有酶活性口袋的组蛋白3赖氨酸9脱甲基酶， 最终被小分子所靶向。值得注意的是，我们的研究还表明，JMJD 1C对于 不仅t（8;21）细胞存活，而且几种不同类型的AML细胞也存活，这表明其作为一种免疫调节剂的潜在作用。 共享关键转录因子的通用共激活因子，为治疗提供了另一个机会。 通过抑制TF-辅因子相互作用来促进发育。 基于我们已发表的工作和初步研究，我们建议继续和扩大我们的原始研究。 建议在以下几个方面进行研究。首先，我们将系统地剖析新指示的子复合体 的AETFC，并确定他们如何指导辅激活子和辅阻遏物的重要靶基因（目的1）。 其次，我们将详细介绍不同类型的pan-JMJD 1C依赖性的分子机制。 白血病（Aim 2）。我们将采用几种互补的方法，包括：（i）无细胞系统 用纯化的因子和DNA/染色质模板重建，以揭示直接的辅因子效应， （ii）基于白血病细胞的测定，以监测引入突变体后的动态变化，或 删除TF网络的组件;（iii）全基因组分析，包括ATAC-seq，ChIP-seq和 RNA测序第三，我们将开发/采用新的t（8;21）白血病小鼠模型，以验证新发现的 AML中的参与者/互动（目标3）。总而言之，所提出的方法将挖掘出更多的机制 新的见解和蛋白质/酶靶点，将为AML提供新的治疗机会。

PROJECT SUMMARY Development is controlled by the timely expression and function of transcription factors that regulate gene expression and determine cell fate and function. Leukemia is often driven by chromosomal translocations that result in the production of novel transcription factors, produced by the abnormal fusion of two separate gene products. In acute myelogenous leukemia (AML), the most common such fusion is the t(8;21)-derived AML1- ETO (AE), which drives leukemia by co-opting additional normal cellular transcription factors that are critical for blood stem cell self-renewal and proliferation, thereby trapping the cells in a proliferative immature state that is the basis of leukemia. It has proved difficult to inhibit transcription factor functions pharmacologically, leading us to look for essential AE cofactors that are more tractable drug targets. Our goal is to combine different experimental approaches to decipher the mechanisms of gene regulation in AML, including identification of new players in different hierarchies of regulation that will reveal new opportunities for therapeutics. In recent studies, we identified an AE-associated transcription factor/cofactor complex (AETFC) and pinpointed a novel interaction surface between AE and E proteins that is critical for AETFC function and leukemogenesis. Most importantly, we showed in mouse models that disruption of this interaction severely reduces AE oncogenicity. In addition, we also identified JMJD1C as a novel coactivator for AETFC. JMJD1C is of special interest in that it is a histone 3 lysine 9 demethylase with an enzymatic activity pocket that can ultimately be targeted by small molecules. Notably, our studies have also shown that JMJD1C is critical for survival not only of t(8;21) cells, but also several different types of AML cells, suggesting its potential role as a general coactivator for shared key transcriptional factors, providing another opportunity for therapeutic development through inhibiting TF-cofactor interaction. Based on our published work and preliminary studies, we propose to continue and expand our original proposed studies in the following aspects. First, we will systematically dissect newly indicated sub-complexes of AETFC and determine how they guide coactivators and corepressors to important target genes (Aim 1). Second, we will detail the molecular mechanisms underlying the pan-JMJD1C dependency of different types of leukemia (Aim 2). We will employ several complementary approaches that include: (i) cell-free systems reconstituted with purified factors and DNA/chromatin templates, to reveal direct cofactor effects and mechanisms; (ii) leukemic cell-based assays to monitor dynamic changes upon introduction of mutants or deletion of components of TF networks; (iii) genome-wide analyses that include ATAC-seq, ChIP-seq and RNA-seq. Third, we will develop/employ new mouse models for t(8;21) leukemia to validate newly identified players/interactions in AML (Aim 3). Altogether, the proposed approaches will unearth additional mechanistic insights and protein/enzyme targets that will provide novel therapeutic opportunities in AML.