Project 1 - Genomics of Intermediate-Risk AML Progression and Relapse.

项目摘要/摘要 项目1：中危AML进展和复发的基因组学。这样做的长期目标是 该项目是为了更好地了解有助于疾病进展的遗传和表观遗传事件 和复发的中等风险急性髓细胞白血病患者，并利用他们的治疗。中等风险 急性髓系白血病(最常见的是由DNMT3A突变引发)可以有非常不同的结果，例如 原因仍然不是很清楚。在这个提案中，我们将探索遗传和表观遗传因素 这会影响白血病前期祖先克隆人如何进展为急性髓系白血病(即，进展)，然后演变为 缓解后复发(即“复发”)。几乎所有的AML样本在呈现时都是克隆异质性的， 通常包含从创始克隆(或其他亚克隆)派生的一个或多个亚克隆。亚克隆 通常表现出对治疗的不同敏感性，而耐治疗的亚克隆通常随着新的 在复发时不被认为是驱动因素的突变。以确定表观遗传因素是否也可能 与亚克隆进化相关，我们使用单细胞RNA测序(scRNA- SEQ)，并定义了复发时的转录进化，并检测到复发特异的差异表达 通过批量RNA测序无法检测到的基因。在这个提案中，我们将利用单细胞方法来 更好地了解复发时的亚克隆进化，并评估DNMT3A突变对白血病模式的作用 临床表现和复发时的基因表达。我们还将确定DNMT3A的起始突变是否 仅在创建白血病前期“状态”时需要，或者它们是否也与维持完全 转化的AML细胞。这两个目标的研究可能会改善急性髓细胞白血病的风险评估和治疗： 具体目标1：我们将定义导致克隆进化和复发的事件 中等风险的AML患者。我们将进行增强的全基因组测序(EWGS)和scRNA- 关于中等风险AML样本中匹配的呈现和复发样本的SEQ，这将使我们能够 归因于亚克隆的表达特征，它们在复发时是如何进展的，并识别基因和/或 在显性复发亚克隆中通常不受调控的通路。 具体目标2：我们将确定DNMT3A突变在AML启动和维持中的作用。我们 用可诱导的WT DNMT3A转基因产生了DNMT3A缺陷小鼠(“DNMT3A Null-3A addback”) 可以准确地重新甲基化移植的骨髓细胞的基因组。我们将产生类似的 携带条件性Dnmt3aR878H突变的Addback小鼠，确定它们的DNA甲基化表型，以及 用DNMT3A修复来表征它们的再甲基化动力学和准确性。我们将创造各种不同的 两个模型中的协同突变导致AML，然后确定这些AML是否可以有自己的 恢复DNMT3A的表达改变了细胞的生长和/或分化。这些研究将为临床前提供 将研究针对人类AML细胞DNMT3AR882H突变的药物的效果的试验。

Project Summary/Abstract Project 1: Genomics of intermediate-risk AML progression and relapse. The long-term goal of this project is to better understand the genetic and epigenetic events that contribute to the progression and relapse of patients with intermediate-risk AML, and exploit them therapeutically. Intermediate-risk AML (which is most commonly initiated by DNMT3A mutations) can have vastly different outcomes, for reasons that are still not well understood. In this proposal, we will explore the genetic and epigenetic factors that influence how a pre-leukemic, ancestral clone progresses to AML (i.e. “progression”), and then evolves to recur after a remission (i.e. “relapse”). Virtually all AML samples are clonally heterogeneous at presentation, generally containing one or more subclones derived from a founding clone (or other subclones). Subclones often display different susceptibilities to therapies, and therapy-resistant subclones often evolve with new mutations that are not recognized as drivers at relapse. To determine whether epigenetic factors may also be relevant for subclonal evolution, we have performed pilot studies using single-cell RNA-sequencing (scRNA- seq), and defined transcriptional evolution at relapse, and detected relapse-specific, differentially expressed genes that were not detectable by bulk RNA-sequencing. In this proposal, we will exploit single cell methods to better understand subclonal evolution at relapse, and evaluate the role of DNMT3A mutations for patterns of gene expression at presentation and relapse. We will also determine whether initiating mutations in DNMT3A are required only for creating the preleukemic “state”, or whether they are also relevant for maintaining fully transformed AML cells. The studies of both aims may improve risk assessment and therapeutics for AML: Specific Aim 1: We will define the events that contribute to clonal evolution and relapse in intermediate-risk AML patients. We will perform enhanced whole genome sequencing (eWGS) and scRNA- seq on matched presentation and relapse samples from intermediate-risk AML samples, which will allow us to impute the expression signatures of subclones, how they progress at relapse, and identify genes and/or pathways that are commonly dysregulated in dominant relapse subclones. Specific Aim 2: We will define the role of DNMT3A mutations for AML initiation and maintenance. We have generated Dnmt3a deficient mice with an inducible WT DNMT3A transgene (“Dnmt3a null-3A addback” mice) that can accurately remethylate the genomes of transplanted bone marrow cells. We will generate similar addback mice with a conditional Dnmt3aR878H mutation, define their DNA methylation phenotype, and characterize their remethylation kinetics and accuracy with DNMT3A restoration. We will create a variety of cooperating mutations in both models to cause AML, and then determine whether these AMLs can have their growth and/or differentiation altered by restoring DNMT3A expression. These studies will inform preclinical trials that will study the effects of drugs designed to target the DNMT3AR882H mutation in human AML cells.