Molecular Pathogenesis of Acute Myeloid Leukemia

 描述(由申请人提供)：在这项提案中，我们将尝试确定急性髓系白血病(AML)启动突变的确切分子机制，并将这些机制用于治疗。绝大多数急性髓系白血病患者仍然死于他们的疾病。迫切需要更有效、毒性更低的新疗法。最近的AML基因组测序研究告诉我们，几乎所有的AML肿瘤都是克隆性异质性的。每个肿瘤都起源于一个创始克隆，该克隆是由一种启动突变产生的，该突变允许单个造血干细胞/祖细胞(HSPC)获得克隆优势。这个“白血病前期”克隆获得了额外的、协同的突变，这些突变导致了一个创始克隆的发展，以及临床上明显的AML。亚克隆产生于最初的克隆，也可以从其他亚克隆进化而来。无论如何，所有的亚克隆都包含最初的克隆突变。虽然协作性突变对于靶向治疗(例如，Flt3和/或IDH1/2抑制剂)通常很有吸引力，但它们有时在亚克隆中被发现(即它们只在白血病细胞总数的一小部分中)；亚克隆的治疗性靶向不能被期望是治愈的。这项工作的中心假设是，需要完全了解启动突变的后果，才能充分了解AML的发病机制。我们还假设，针对启动突变的治疗方法最有可能为AML患者提供长期好处。我们将充分描述两个常见的、经过充分验证的AML启动突变(PML-RARA和DNMT3A R882H)，它们都与造血细胞的深刻表观遗传学变化有关。我们将利用最先进的技术(包括全面的、链特异的大小RNA序列、全基因组亚硫酸氢盐测序、染色质可及性研究以及癌基因结合和组蛋白修饰的芯片序列研究)来精确定位这些启动突变的关键基因组靶点，并利用无偏见的蛋白质组学技术来全面识别与突变蛋白特异相互作用的蛋白质。我们将整合这些数据，以确定由启动者改变的基因、RNA、基因座和途径 突变，并就可能与AML发病相关的机制提出新的假设。我们将在人类胚胎干细胞和表达PML-RARA或DNMT3A R882H的转基因小鼠中建立AML启动突变和下游通路的模型，以充分探索通路(例如DNA甲基化和/或组蛋白修饰物)和/或协同突变对它们的作用可能至关重要。作为这项工作的翻译目标，我们将试图开发一种新药，它将抑制突变的DNMT3A R882H蛋白的作用，该蛋白作为WT DNMT3A的显性负抑制物，从而抑制HSPC中的从头DNA甲基化。这种突变导致局部的、典型的DNA低甲基化，这一事件可能被有效的抑制剂逆转，从而可能恢复正常的HSPC功能。

 DESCRIPTION (provided by applicant): In this proposal, we will attempt to determine the precise molecular mechanisms by which acute myeloid leukemia (AML)-initiating mutations act, and to exploit these mechanisms therapeutically. The vast majority of patients who develop AML still die from their disease. New therapies that are more efficacious and less toxic are urgently needed. Recent AML genome sequencing studies have taught us that virtually all AML tumors are clonally heterogeneous. Each tumor originates from a founding clone that was created by an initiating mutation that allowed a single hematopoietic stem/progenitor cell (HSPC) to achieve a clonal advantage. This `preleukemic' clone acquires additional, cooperating mutations that lead to the development of a founding clone, and clinically apparent AML. Subclones arise from the founding clone, or can evolve from other subclones. Regardless, all subclones contain the founding clone mutations. Although cooperating mutations are often attractive for targeted therapies (e.g. FLT3 and/or IDH1/2 inhibitors), they are sometimes found in subclones (i.e. they are only in a fraction of the total leukemic cell population); therapeutic targeting of subclones cannot be expected to be curative. The central hypothesis of this work is that a complete understanding of the consequences of initiating mutations is required to fully understand AML pathogenesis. We also hypothesize that therapeutic approaches directed against initiating mutations are the most likely to provide long-term benefit for AML patients. We will fully characterize two common, well-validated AML-initiating mutations (PML-RARA and DNMT3A R882H) that are both associated with profound epigenetic alterations in hematopoietic cells. We will utilize state-of-the-art techniques (including comprehensive, strand-specific RNA-seq of large and small RNAs, whole genome bisulfite sequencing, chromatin accessibility studies, and ChIP-seq studies for oncogene binding and histone modifications) to pinpoint the key genomic targets of these initiating mutations, and unbiased proteomic techniques to comprehensively identify proteins that interact specifically with the mutant proteins. We will integrate these data to identify genes, RNAs, loci, and pathways that are altered by the initiating mutations, and develop new hypotheses regarding mechanisms that may be relevant for AML pathogenesis. We will model AML-initiating mutations and downstream pathways both in human embryonic stem cells, and in transgenic mice expressing PML-RARA or DNMT3A R882H, to fully explore the contributions of pathways (e.g. DNA methylation and/or histone modifiers) and/or cooperating mutations that may be critical for their actions. As a translational goal of thi work, we will attempt to develop a novel drug that will inhibit the action of the mutant DNMT3A R882H protein, which acts as a dominant negative inhibitor of WT DNMT3A, thereby suppressing de novo DNA methylation in HSPCs. This mutation causes in focal, canonical, DNA hypomethylation, an event that may be reversed by an effective inhibitor, which may restore normal HSPC function.