DNMT3A in Development of Hematologic Malignancies

摘要 DNA甲基转移酶3A(DNMT3A)是近8年来出现的一种 造血系统中最重要的肿瘤抑制因子，在大多数类型中发生突变 人类恶性血液病，在超过20%的急性髓系白血病中发现 急性淋巴细胞白血病(AML)以及急性淋巴细胞性白血病(ALL)和淋巴瘤。通过机制， DNMT3A突变被认为为继发性疾病提供了肥沃的土壤 导致坦率的恶性行为的突变。在上一个资助期，我们试图建立 并利用识别的DNMT3A相关恶性肿瘤研究可靠的肿瘤模型 DNMT3A突变与Flt3基因内部串联复制(ITD)的协同作用 导致髓系和淋巴系类型的高度穿透性恶性肿瘤。在这里，我们将学习 最早的事件代表了从克隆性造血向恶性转化的转变。我们 假设DNMT3A突变和NPM1突变通过实施 互补的表观遗传学变化，用于维持突变的细胞处于类似于HSC的状态。我们 预计这种失调的一个关键影响是HOX基因的异常表达，这种基因驱动着自我 更新。我们将在这里使用小鼠模型剖析这种情况发生的机制， 人类细胞系和人类原生样本。我们的长期目标是利用发展出来的洞察力 在这里加强差异化和开发新的治疗策略。我们将(1)确定 表观遗传学和分子变化与恶性肿瘤的发展有关 DNMT3A缺陷的造血祖细胞。使用具有DNMT3A-KO突变等位基因的小鼠 和诱导的NPM1c，我们将研究在表观遗传和 癌前干细胞和前体细胞的转录水平。(2)检查依赖关系 具有突变的DNMT3A、NPM1和Flt3-ITD的AML。我们假设这种常见的子类型 AML依赖于特定基因的持续表达，如Hox和Meis1。我们 将使用CRISPR KO或目标DNA检查此依赖项和其他潜在依赖项 甲基化。(3)检测人DNMT3A突变的急性髓系白血病细胞表观基因组重塑和 依赖关系。我们将验证目标1和目标2中确定的目标，并探索特定目标的价值 调节因子，如NPM1的核重新定位，DNMT3A突变等位基因的纠正， 以及特定靶点的重新甲基化。这些研究将揭示逐步的表观基因组学 由于丢失DNMT3A而导致的AML以及它们的一些 依赖关系。这将有助于更好地理解DNMT3A的丢失是如何促进 由于发现了新的靶点，可能会导致新的治疗策略。

Abstract DNA METHYLTRANSFERASE 3A (DNMT3A) has emerged over the past ~8 years as one of the most important tumor suppressors in the hematopoietic system, being mutated across most types of human hematologic malignancies, and found in greater than 20% of acute myeloid leukemias (AMLs) as well as acute lymphoid leukemias (ALLs) and lymphomas. Through mechanisms that are not understood, DNMT3A mutations are thought to provide a fertile ground for secondary mutations which drive the frank malignancy. In the previous funding period, we sought to establish and study a reliable tumor model of DNMT3A-associated malignancies using the recognized collaboration between DNMT3A-mutation and the internal tandem duplication (ITD) of FLT3 which results in highly penetrant malignancies of both myeloid and lymphoid types. Here, we will study the very earliest events that represent the transition from clonal hematopoiesis to malignacy. We hypothesize that DNMT3A mutations and NPM1 mutations collaborate effectively by enforcing complementary epigenetic changes that serve to maintain mutated cells in an HSC-like state. We expect that a key effect of this dysregulation is aberrant expression of HOX genes that drives self- renewal. We will dissect the mechanisms through which this occurs here using mouse models, human cell lines, and human primary samples. Our long-term goal is to use insights developed here to enforce differentiation and develop new therapeutic strategies. We will (1) Identify the epigenetic and molecular changes associated with the development of malignancies from Dnmt3a-deficient hematopoietic progenitors. Using mice that have mutant alleles of Dnmt3a-KO and inducible NPM1c, we will examine the concerted changes that occur at the epigenetic and transcriptional levels in pre-malignant stem and progenitor cells. (2) Examine the dependencies of AML with mutated DNMT3A, NPM1, and FLT3-ITD. We hypothesize this common sub-type of AML is dependent on the sustained expression of particular genes such as Hox and Meis1. We will examine this and other potential dependencies using CRISPR KO or targeted DNA methylation. (3) Examine in human DNMT3A-mutated AML cells epigenome remodeling and dependencies. We will validate targets identified in Aims 1 and 2, and explore the value of specific modulators such as nuclear re- localization of NPM1, correction of the DNMT3A-mutant allele, and re-methylation of specific target sites. These studies will reveal the stepwise epigenomic changes that occur due to loss of DNMT3A that lead to AML as well as some of their dependencies. This will lead to an improved understanding of how loss of DNMT3A promotes malignancies, and potentially to new therapeutic strategies due to identification of new targets.