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

项目总结/摘要 项目1：中危AML进展和复发的基因组学。长期目标是 该项目旨在更好地了解有助于进展的遗传和表观遗传事件 和复发的中度风险AML患者，并在治疗上利用它们。中危 AML（最常见的是由DNMT 3A突变引发）可能会有截然不同的结果， 原因还不太清楚。在这个建议中，我们将探讨遗传和表观遗传因素 影响白血病前的祖先克隆如何进展为AML（即“进展”），然后演变为 缓解后复发（即“复发”）。几乎所有AML样品在呈现时都是克隆异质性的， 通常含有一个或多个衍生自基础克隆（或其它亚克隆）的亚克隆。亚克隆 通常表现出对治疗的不同敏感性，并且治疗抗性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。为了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），并定义复发时的转录进化，并检测复发特异性差异表达的 通过批量RNA测序检测不到的基因。在本提案中，我们将利用单细胞方法， 更好地了解复发时的亚克隆进化，并评估DNMT 3A突变对复发模式的作用。 在发病和复发时的基因表达。我们还将确定启动DNMT 3A突变是否 是否仅仅是创造白血病前期“状态”所必需的，或者它们是否也与维持完全的 转化的AML细胞。这两个目标的研究可能会改善AML的风险评估和治疗： 具体目标1：我们将定义有助于克隆进化和复发的事件， 中危AML患者。我们将进行增强型全基因组测序（eWGS）和scRNA- seq对来自中等风险AML样本的匹配表现和复发样本进行测序，这将使我们能够 估算亚克隆的表达特征，它们在复发时如何进展，并鉴定基因和/或 在显性复发亚克隆中通常失调的途径。 具体目标2：我们将确定DNMT 3A突变在AML启动和维持中的作用。我们 已经产生了具有可诱导的WT DNMT 3A转基因的Dnmt 3a缺陷小鼠（“Dnmt 3a无效-3A加回”）， 小鼠），其可以精确地使移植的骨髓细胞的基因组再甲基化。我们将产生类似的 具有条件性Dnmt 3aR 878 H突变的addback小鼠，定义其DNA甲基化表型，以及 用DNMT 3A恢复来表征它们的再甲基化动力学和准确性。我们将创建各种 在两种模型中协同突变导致AML，然后确定这些AML是否可以具有其 通过恢复DNMT 3A表达改变生长和/或分化。这些研究将告知临床前 这些试验将研究旨在靶向人类AML细胞中DNMT 3AR 882 H突变的药物的作用。

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.