Characterising and targeting aberrant enhancer function in acute myeloid leukaemia (AML)

急性髓性白血病（AML）是一种侵袭性血液学癌症。它是欧洲最常见的急性白血病，发病率为每10万人中3-4人。此外，其发病率随着年龄的增长而上升，因此随着总人口的老龄化，它将成为一个越来越普遍的问题。AML的总体生存率低于30%，在过去的25年里，主要的治疗方法是积极的、有毒的联合化疗，表面上没有变化。因此，迫切需要新的治疗方法来改善AML的治疗效果。在对疾病机制有了更深入了解的地方，往往会取得治疗方面的成果。最令人印象深刻的例子是急性早幼粒细胞白血病亚型，对这种亚型的机制的理解导致了新疗法（全反式氨基酸、ATRA和三氧化二砷）的发展，可以治愈高达90%的患者。因此，更好地了解AML生物学是开发新疗法以改善治疗效果的先决条件。最近，许多研究证明AML与基因的异常表达有关，我们和其他人已经表明，使用小分子抑制剂等药物纠正这些异常基因程序可能是一种有希望的治疗方法。基因的表达在身体的不同组织之间差异很大，这些差异是由称为增强子的DNA元件调节的。增强子以组织特异性的方式通过与启动子（基因的DNA起始位点）的通信来调节基因的表达，因此对于一个基因X，可能有许多不同的增强子在不同的组织（如血液、大脑和皮肤）中调节X的表达。增强子通常与启动子相距很远，需要通过形成DNA环与启动子进行三维接触。增强子的功能反过来由称为转录因子的分子和染色质（DNA及其蛋白质支架）的调节因子控制和模拟。最近的研究表明，增强子、转录因子（TF）和染色质调节因子在AML中都是经常性和常见的突变。因此，我们提出异常增强子功能是AML的病因，并且有可能在治疗上靶向这种异常功能来纠正异常基因表达程序，从而关闭白血病。本提案将解决AML与正常造血（正常血液形成过程）进化过程中增强剂的使用和功能的差异。为了让我们有前瞻性地建立模型，我们将使用具有不同aml特异性突变组合的小鼠模型来模拟疾病的不同阶段：正常、白血病前期和坦率地说，白血病阶段。我们将使用一种称为ChIP-Seq的技术，将特定蛋白质与DNA结合，比较不同阶段增强子的存在、使用和功能。此外，使用另一种称为Hi-C的尖端技术，我们将绘制增强子和启动子之间的循环相互作用，以及它们在疾病阶段的差异。我们还将测量最终输出，即基因的表达，详细说明阶段之间的差异以及这些差异如何与增强子的使用相关。然后，我们将这些模式与携带相同突变的人类白血病进行比较。这些数据将使我们能够确定控制白血病特异性增强子的TF和染色质调节因子。我们的最后一个目标将是抑制这些因素，作为一个概念的证明，增强功能可能是一个治疗目标。我们将使用类似药物的小分子抑制剂和遗传技术来改变这些调节因子的功能，并将在试管实验和白血病活体模型中寻找白血病生长和基因表达的改变。

Acute myeloid leukaemia (AML) is an aggressive haematological cancer. It is the most common acute leukaemia in Europe, with an incidence of 3-4 per 100,000 of the population. In addition, its incidence rises with age, therefore it will become an increasingly common problem as the general population ages. AML has a dismal overall survival rate of less than 30% and the mainstay of treatment aggressive, toxic, combination chemotherapy has remained ostensibly unchanged for the last 25 years. Novel therapies are therefore urgently required to improve treatment outcomes in AML. Where a greater knowledge of the mechanisms of disease have been uncovered, therapeutic gains have often been achieved. The most impressive example of this is the acute promyelocytic leukaemia subtype, where an understanding of the mechanisms that underlie this subtype has led to the development of novel therapies (All Trans Retionoic Acid, ATRA and arsenic trioxide) that can cure up to 90% of patients. Therefore, a better understanding of AML biology is a pre-requisite for the development of novel therapies to improve treatment outcomes.Recently, a number of studies have documented that AML is associated with abnormal expression of genes and we and others have shown that correction of these abnormal gene programmes, with drugs such as small molecule inhibitors, may be a promising therapy. The expression of genes varies greatly between different tissues in the body and these differences are regulated by DNA elements called enhancers. Enhancers regulate the expression of genes by communication with promoters (the DNA start sites of genes) in a tissue-specific manner, such that for a gene X, there may be a number of different enhancers that regulate the expression of X in different tissues such as blood, brain and skin. Enhancers are a often a long distance away from promoters and require 3-dimensional contact with promoters through the formation of DNA loops. The function of enhancers is in turn controlled and modelled by molecules called transcription factors and regulators of chromatin (DNA and its protein scaffold). Recent work has shown that enhancers, transcription factors (TF) and chromatin regulators are all recurrently and commonly mutated in AML. We therefore propose that abnormal enhancer function is causative in AML and that it may be possible to therapeutically target this abnormal function to correct abnormal gene expression programmes thereby switching off leukaemia.This proposal will address how enhancer usage and function differ during the evolution of AML from normal haematopoiesis, the process of normal blood formation. To allow us to model this prospectively, we will use mouse models with different combinations of AML-specific mutations that mimic different stages of the disease: normal, pre-leukaemic and frankly leukaemia stages. We will compare the presence, usage and function of enhancers between stages using the binding of specific proteins to DNA, a technique called ChIP-Seq. In addition, using another cutting-edge technique called Hi-C, we will map looping interactions between enhancers and promoters and how these differ by stage of disease. We will also measure the end output, that is expression of genes, detailing differences between the stages and how these correlate with enhancer usage. We will then compare these patterns to human leukaemia's that carry the same mutations. These data will allow us to identify the TF and chromatin regulators that control leukaemia specific enhancers. Our last objective will be to inhibit these factors, as a proof of concept that enhancer function may be a therapeutic target. We will use drug-like small molecular inhibitors and genetic techniques to alter the function of these regulators and will look for alterations of leukaemia growth and gene expression both in test tube experiments and live models of leukaemia.