Molecular Pathogenesis of Acute Myeloid Leukemia

急性髓系白血病的分子发病机制

• 批准号: 10678908
• 负责人: TIMOTHY J. LEY
• 金额: $ 91.43万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-13 至 2029-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678908
• 关键词: ATAC-seq; Acute Myelocytic Leukemia; Back; CBFA2T1 gene; CBFbeta-MYH11 fusion protein; Cell model; Cells; ChIP-seq; Clinical Trials Design; Communities; Core-Binding Factor; DNA; DNA sequencing; DNMT3B gene; DNMT3a; DNMT3a mutation; Data Analyses; Data Set; Databases; Development; Disease; Epigenetic Process; Event; Funding; Genetically Engineered Mouse; Genomics; Goals; Growth; HDAC1 gene; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Histones; Human; Methods; Molecular; Mutation; PML-RARalpha protein; Pathogenesis; Patients; Pharmaceutical Preparations; Precision therapeutics; Preleukemia; Process; Protein Isoforms; Proteins; Proteomics; RUNX1 gene; Sampling; Series; Work; acute myeloid leukemia cell; bisulfite sequencing; blastomere structure; embryo cell; engineered stem cells; epigenomics; fitness; genetic approach; genomic locus; induced pluripotent stem cell; inhibitor; insight; leukemia initiating cell; mouse model; mutant; novel; overexpression; phosphoproteomics; programs; proteogenomics; single cell technology; targeted treatment; therapy design; transcriptome sequencing; whole genome

Project Summary The long-term goal of this project is to develop effective, precision therapies directed against the initiating mutations of Acute Myeloid Leukemia (AML). During the current funding period, we performed a series of genomic and epigenomic studies that have clarified the mechanisms that AML initiating mutations use to "reprogram" hematopoietic stem progenitor cells (HSPCs), increasing their fitness for transformation. In the next funding period, we will use primary human AML samples, induced pluripotent stem cells (iPSCs), and genetically engineered mouse models to further evaluate the molecular mechanisms involved in preleukemic reprogramming, and progression to AML. Four well characterized events (that initiate more than half of AML cases) will be studied in detail. We will continue our work with DNMT3A mutations and PML- RARA, and add the study of Core Binding Factor AML fusions (RUNX1-RUNX1T1 and CBFB-MYH11). The "toolkit" for these studies will involve the analysis of preleukemic and fully transformed hematopoietic cells from these models, using bulk DNA and RNA sequencing, whole genome bisulfite sequencing, ATAC-seq, ChIP- seq and/or CUT&RUN to detect the genomic locations of activating and repressive histone marks (and the fusions themselves), and single cell technologies for RNA, DNA, and ATAC-seq. We will also be performing comprehensive proteomic studies to complete "proteogenomic" datasets for these initiating events, including 1) the identification of the hematopoietic proteins that interact with the initiating proteins listed above, and 2) the development of quantitative deep-scale proteomic and phosphoproteomic datasets broadly representative of all AML subtypes. The integrative analysis of these datasets (and their availability to the AML community) should provide important new insights about AML pathogenesis, and suggest mechanistically targeted therapies. In this proposal, we provide one representative example of this process: using novel methods to identify proteins that interact with DNMT3A, we discovered several mutations that disrupt the normal interaction of DNMT3A with an inactive isoform of DNMT3B (DNMT3B3); these mutations destabilize DNMT3A and decrease its activity. Remarkably, we found that we can restore the activity of many mutant DNMT3A proteins by retrovirally overexpressing DNMT3L, a protein that normally interacts with DNMT3A and 3B in embryonic cells to increase their activity. "Addback" of DNMT3L into hematopoietic cells with the Dnmt3aR878H mutation remethylates DNA, and decreases the growth of AML cells initiated by this mutation. Since DNMT3L is epigenetically silenced in nearly all AMLs, a program to identify drugs and genetic strategies to reactivate DNMT3L in AML cells will be developed. We have already found that Romidepsin, an HDAC1 inhibitor, potently induces DNMT3L expression, and a clinical trial designed to evaluate the activity of this drug in DNMT3A mutant AMLs is planned. Additional approaches for developing mechanistically driven therapies designed to thwart initiating mutations will be developed during the next funding period.

项目概要 该项目的长期目标是开发针对该疾病的有效、精准疗法 急性髓系白血病（AML）的起始突变。在本轮融资期间，我们进行了 一系列基因组和表观基因组研究阐明了 AML 启动突变的机制 “重新编程”造血干祖细胞（HSPC），提高它们的转化适应性。在 下一个资助期，我们将使用原始人类 AML 样本、诱导多能干细胞 (iPSC)、 和基因工程小鼠模型以进一步评估涉及的分子机制 白血病前期重编程和进展为 AML。四个特征明确的事件（引发更多 超过一半的 AML 病例）将得到详细研究。我们将继续研究 DNMT3A 突变和 PML- RARA，并添加核心结合因子AML融合体（RUNX1-RUNX1T1和CBFB-MYH11）的研究。这 这些研究的“工具包”将涉及对来自白血病前期和完全转化的造血细胞的分析 这些模型使用大量 DNA 和 RNA 测序、全基因组亚硫酸氢盐测序、ATAC-seq、ChIP- seq 和/或 CUT&RUN 来检测激活和抑制组蛋白标记的基因组位置（以及 融合本身），以及 RNA、DNA 和 ATAC-seq 的单细胞技术。我们还将表演 全面的蛋白质组学研究，以完成这些起始事件的“蛋白质组学”数据集， 包括 1) 鉴定与起始蛋白相互作用的造血蛋白 上面列出的，以及2）定量深度蛋白质组学和磷酸蛋白质组学的发展 数据集广泛代表所有 AML 亚型。这些数据集（及其 AML 社区的可用性）应提供有关 AML 发病机制的重要新见解，并且 建议机械靶向治疗。在本提案中，我们提供了一个具有代表性的示例 过程：使用新方法来识别与 DNMT3A 相互作用的蛋白质，我们发现了几个突变 破坏 DNMT3A 与无活性的 DNMT3B (DNMT3B3) 亚型的正常相互作用；这些 突变会破坏 DNMT3A 的稳定性并降低其活性。值得注意的是，我们发现我们可以恢复活动 通过逆转录病毒过表达 DNMT3L（一种通常相互作用的蛋白质）来产生许多突变的 DNMT3A 蛋白质 与胚胎细胞中的 DNMT3A 和 3B 结合以增加其活性。 DNMT3L“Addback”进入造血系统 具有 Dnmt3aR878H 突变的细胞使 DNA 重新甲基化，并减少由此引发的 AML 细胞的生长 突变。由于 DNMT3L 在几乎所有 AML 中都处于表观遗传沉默状态，因此需要开展一项识别药物和基因的程序 将开发在 AML 细胞中重新激活 DNMT3L 的策略。我们已经发现罗米地辛是一种 HDAC1 抑制剂，有效诱导 DNMT3L 表达，一项旨在评估其活性的临床试验 该药物计划用于 DNMT3A 突变型 AML。开发机械驱动的其他方法 旨在阻止起始突变的疗法将在下一个资助期内开发。

项目成果

Melanoma in a patient with DNMT3A overgrowth syndrome.

• DOI: 10.1101/mcs.a006267
• 发表时间: 2023-04
• 期刊: COLD SPRING HARBOR MOLECULAR CASE STUDIES
• 影响因子: 1.8
• 作者: Chen, David Y.;Sutton, Leslie A.;Ramakrishnan, Sai Mukund;Duncavage, Eric J.;Heath, Sharon E.;Compton, Leigh A.;Miller, Christopher A.;Ley, Timothy J.
• 通讯作者: Ley, Timothy J.