Molecular Pathogenesis of Acute Myeloid Leukemia

项目摘要 这个项目的长期目标是开发有效的、精确的治疗方法，针对 急性髓系白血病(AML)的启动突变。在本资助期内，我们进行了 一系列基因组和表观基因组学研究，阐明了AML启动突变所使用的机制 对造血干细胞(HSPC)进行“重新编程”，增加其转化的适宜性。在 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(IPSCs)、 和基因工程小鼠模型，以进一步评估参与 白血病前期重编程，并进展为急性髓系白血病。四个特征良好的事件(引发更多 超过一半的急性髓系白血病病例)将得到详细研究。我们将继续研究DNMT3A突变和PML- Rara，并增加了核心结合因子AML融合研究(RUNX1-Runx1t1和Cbfb-MYH11)。这个 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- SEQ和/或Cut&Run以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，以完成这些启动事件的“蛋白质基因组”数据集， 包括1)与启动蛋白相互作用的造血蛋白的鉴定 2)定量深层次蛋白质组和磷蛋白质组的发展 广泛代表所有AML子类型的数据集。对这些数据集的综合分析(及其 对AML社区的可用性)应该提供关于AML发病机制的重要新见解，以及 建议进行机械性靶向治疗。在本提案中，我们提供了一个具有代表性的例子 过程：使用新的方法鉴定与DNMT3A相互作用的蛋白质，我们发现了几个突变 扰乱DNMT3A与DNMT3B的非活性亚型(DNMT3B3)的正常相互作用；这些 突变会破坏DNMT3A的稳定性，降低其活性。值得注意的是，我们发现我们可以恢复活动 许多突变的DNMT3A蛋白通过逆转录病毒过表达DNMT3L，这是一种通常相互作用的蛋白质 在胚胎细胞中加入DNMT3A和3B，以提高其活性。DNMT3L进入造血细胞的“回补” 带有Dnmt3aR878H突变的细胞重新甲基化DNA，并减少由此引发的AML细胞的生长 突变。由于DNMT3L在几乎所有的AML中都是表观遗传学沉默的，一个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Romidessin，一种 HDAC1抑制剂，有效地诱导DNMT3L表达，以及一项旨在评估其活性的临床试验 这种药物正在计划用于DNMT3A突变的AML。开发机械驱动的其他方法 旨在阻止启动突变的疗法将在下一个资助期开发。

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.