Molecular Pathogenesis of Acute Myeloid Leukemia

项目摘要 该项目的长期目标是开发有效的、精确的治疗方法， 急性髓性白血病（AML）的起始突变。在当前融资期间，我们执行了一项 一系列基因组和表观基因组研究，阐明了AML起始突变使用的机制 “重新编程”造血干祖细胞（HSPCs），增加其转化适应性。在 下一个资助期，我们将使用原代人类AML样本，诱导多能干细胞（iPSC）， 和基因工程小鼠模型，以进一步评估参与的分子机制， 白血病前重编程和进展为AML。四个特征鲜明的事件（引发更多 超过一半的AML病例）将进行详细研究。我们将继续研究DNMT 3A突变和PML- RARA，并增加核心结合因子AML融合体（RUNX 1-RUNX 1 T1和CBFB-MYH 11）的研究。的 这些研究的“工具包”将包括分析白血病前期和完全转化的造血细胞， 这些模型，使用批量DNA和RNA测序，全基因组亚硫酸氢盐测序，ATAC-seq，ChIP- seq和/或CUT&RUN来检测激活和抑制性组蛋白标记的基因组位置（以及 融合本身），以及用于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 2）定量深层次蛋白质组学和磷酸化蛋白质组学的发展 广泛代表所有AML亚型的数据集。对这些数据集的综合分析（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， 提出了机械靶向治疗。在本提案中，我们提供了一个具有代表性的例子， 过程：使用新的方法来鉴定与DNMT 3A相互作用的蛋白质，我们发现了几个突变 破坏DNMT 3A与DNMT 3B的非活性同种型（DNMT 3B 3）的正常相互作用;这些 突变使DNMT 3A不稳定并降低其活性。值得注意的是，我们发现我们可以恢复 通过逆转录病毒过表达DNMT 3L，许多突变DNMT 3A蛋白，DNMT 3L是一种通常相互作用的蛋白， 与胚胎细胞中的DNMT 3A和3B结合以增加其活性。DNMT 3L“加回”造血细胞 具有Dnmt 3aR 878 H突变的细胞使DNA重新甲基化，并降低由此引发的AML细胞的生长。 突变由于DNMT 3L在几乎所有AML中表观遗传学沉默，因此鉴定药物和遗传学的程序 将开发在AML细胞中重新激活DNMT 3L的策略。我们已经发现，罗米地辛， HDAC 1抑制剂，有效诱导DNMT 3L表达，一项旨在评估HDAC 1抑制剂活性的临床试验 该药物在DNMT 3A突变AML中的应用正在计划中。开发机械驱动的其他方法 在下一个供资期间，将开发旨在阻止初始突变的疗法。

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.