The Role of MYC Mutations in acute Myeloid Leukemia

项目摘要 该提案概述了一项为期5年的研究和职业发展计划，旨在支持候选人的 走向独立的学术生涯的轨迹。拟议的研究项目，重点是急性 髓系白血病(AML)相关的MYC突变及其在AML细胞中的关键靶基因，将利用 关于华盛顿大学医学院可用的关键专业知识和资源。职业生涯 发展计划和教学工作将为应聘者提供各种技能，使其能够成功 向独立过渡。这一建议是基于我们最近的观察，即一套独特的经常性 MYC错义突变在正常核型AML患者的显性克隆中显著丰富 只接受化疗的首次缓解时间很长(5年)；在我们的系列中，6/6 MYC突变与 与NPM1突变(NPMc)一起发生，表明了一种独特的合作形式。根据我们的初步调查 发现，我们假设：1)过度表达WT蛋白或突变的MYC蛋白的AML在生物学上是不同的； 每个MYC突变可能对其转录靶标有独特的影响；3)MYC突变可能相互作用 有NPMc突变。为了解决这些假设，我们提出了以下具体目标： 具体目标1：我们将单独或协同研究AML相关MYC突变的功能 使用NPMc。我们已经开发了一种多西环素诱导的系统，允许我们调节Myc的表达 存在或不包含Npmc突变的造血干细胞和祖细胞(HSPC)。使用这个 系统，我们将评估WT或突变Myc，无论有或没有Npmc，如何影响AML的发展，进展， 以及在体外和体内的AraC反应性。我们还在开发一种更具生理学、条件性的 MYCT58N(AML和B细胞恶性肿瘤中的一种反复发生的突变)的敲入模型将使我们能够 探讨突变型Myc和Npmc的生理剂量对AML发病机制和AraC敏感性的影响。 具体目标2：我们将确定wt与突变体的转录输出和基因组结合位点 WT与Npmc小鼠AML中的MYC蛋白。为了鉴定Myc基因异常，我们将使用scRNA- SEQ以确定由WT与突变体Myc Over启动的AML(在Aim 1中获得)的转录图谱。 表达式，单独或与Npmc结合使用。我们还将进行芯片序列研究，以表征 WT与突变Myc的基因组结合位点，并对这些数据集进行综合分析，以定义 Myc的直接效应与间接效应对AML细胞转录输出的相对贡献。通过组合 功能研究的结果(目标1)与表达和DNA结合研究(目标2)，我们希望 更好地了解Myc突变如何改变造血细胞的转录，以及Npmc如何影响这一输出。 如果这些研究成功，它们可能会为MYC介导的转化提供重要的见解，完善我们的 目前用于AML风险分层的预测工具，确定与AraC敏感性有关的途径，并允许我们 创造治疗急性髓系白血病患者的新方法。

Project Summary This proposal outlines a 5-year research and career development plan designed to support the candidate’s trajectory towards an independent academic career. The proposed research project, which focuses on acute myeloid leukemia (AML)-associated MYC mutations and their key target genes in AML cells, will capitalize on critical expertise and resources available at Washington University School of Medicine. The career development plan and didactic work will provide the candidate with a variety of skills that will enable a successful transition to independence. This proposal is founded on our recent observation that a unique set of recurrent MYC missense mutations are significantly enriched in the dominant clones of normal karyotype AML patients who have very long first remissions (>5 years) with chemotherapy only; in our series, 6/6 MYC mutations co- occurred with NPM1 mutations (NPMc), suggesting a unique form of cooperativity. Based on our preliminary findings, we hypothesize that: 1) AMLs overexpressing WT or mutant MYC proteins are biologically different; 2) each MYC mutation may have unique effects on its transcriptional targets; and 3) MYC mutations may cooperate with NPMc mutations. To address these hypotheses, we propose the following specific aims: Specific Aim 1: We will examine the function of AML-associated MYC mutations alone, or in cooperation with NPMc. We have developed a doxycycline-inducible system that allows us to regulate Myc expression in hematopoietic stem and progenitor cells (HSPCs) that do or do not contain the Npmc mutation. Using this system, we will evaluate how WT or mutant Myc, with or without Npmc, impacts AML development, progression, and AraC responsiveness both in vitro and in vivo. We are also developing a more physiologic, conditional knock-in model of MYCT58N (a recurrent mutation both in AML and B cell malignancies) that will allow us to explore the influence of physiologic doses of mutant Myc and Npmc on AML pathogenesis and AraC sensitivity. Specific Aim 2: We will define the transcriptional outputs and genomic binding sites of WT vs. mutant Myc proteins in AMLs arising in WT vs. Npmc mice. To identify Myc dysregulated genes, we will use scRNA- seq to determine the transcriptional profiles of AMLs (obtained in Aim 1) initiated by WT vs. mutant Myc over- expression, either alone or in combination with Npmc. We will also perform ChIP-seq studies to characterize the genomic binding sites of WT vs. mutant Myc, and perform an integrated analysis of these datasets to define the relative contributions of direct vs. indirect effects of Myc on transcriptional outputs in AML cells. By combining the results of the functional studies (Aim 1) with the expression and DNA binding studies (Aim 2), we hope to better understand how Myc mutations alter transcription in hematopoietic cells, and how Npmc affects this output. If these studies are successful, they may provide important insights on MYC-mediated transformation, refine our current predictive tools for AML risk stratification, identify pathways involved in AraC sensitivity, and allow us to create new approaches for the treatment of AML patients.