The role of the histone chaperone Chaf1b in sustaining the Hoxa9-driven AML differentiation block

项目摘要/摘要 我在康奈尔大学获得本科学位，在普林斯顿大学获得分子生物学博士学位 目前是白血病和淋巴瘤的博士后研究员和特别研究员 哈佛医学院和波士顿儿童医院的社会。在研究了分子基础之后 在读研究生时，我过渡到了杨的实验室的表观遗传学领域 她是我的博士后。我在博士后期间接受了广泛的实验和学术培训。我学到了 基础染色质生物化学的严谨性是我的第一个博士后项目，最近发表了第一个合著者 论文就是在此基础上展开的。这项研究作为封面文章出现在《细胞》杂志上，并报道了 一种新的后生动物表观遗传DNA修饰N6-甲基腺嘌呤(6 MA)的鉴定。我的学问 培训涉及广泛的科学写作，使我能够写出三个成功的博士后 奖学金申请(来自NIH、ACS和LLS)，基于我的工作为史博士提供的两项成功的资助 (来自塞缪尔·韦克斯曼癌症研究基金会和哈佛表观遗传学倡议)，以及一 最近作为合著者在《自然评论分子生物学》杂志上发表了评论论文。 我目前的博士后研究将我在癌症生物学方面的背景与我最近接受的 染色质生物化学，重点是急性分化障碍的表观遗传学基础 髓系白血病(AML)。急性髓系白血病是最致命的血液系统恶性肿瘤，其原因超过 美国每年有10,000人死亡。AML通常采用化疗治疗，尽管患者经常复发并患有 治疗选择有限。早幼粒细胞亚型AML可通过“分化疗法”治愈-- 维甲酸诱导分化和抑制增殖--但这种方法 在其他AML亚型中无效。最近的研究表明，这种对非APL AML分化的阻断 在本质上是表观遗传的。稳定但可逆的染色质改变被认为使髓系丧失能力 这些细胞中的分化基因表达程序。我最近建立了一种强大的基因筛查 揭示非APL AML分化计划的表观遗传调控因素的方法。这些屏幕上有 发现Caf-1组蛋白重塑复合体的Chaf1b亚单位是Hoxa9的关键调节因子。 驱动急性髓系白血病分化阻断。本申请中提出的工作的总体目标是确定 Chaf1b调节AML细胞分化的基于染色质的分子机制。 我在这个提案中的第一个目标是使用生化方法来鉴定蛋白质和长时间的非 编码与Chaf1b和AML细胞中更大的Caf-1复合体相关的RNA(LncRNAs)。那我会的 重点确定这些相互作用的蛋白质和RNA中哪些对招募Chaf1b到ITS最关键 靶向基因组位置，并帮助维持其作为分化阻断蛋白的活性。在《目标2》中，我将使用 广泛的表观基因组图谱方法识别染色质重塑和转录过程 随着细胞分化对Chaf1b的抑制，这些基因会发生改变。然后我会比较这些表观基因组 从我最近对正常的、生理性的髓系分化程序的描述中获得的数据 确定Chaf1b的缺失是否通过典型或非典型机制诱导分化。最后，在 目的通过检测Chaf1b的抑制作用，确定其作为候选治疗靶点的潜力。 诱导小鼠急性髓系白血病模型分化，减轻疾病负担或影响正常造血。 虽然我目前是博士后，但拟议的研究将作为独立调查人员进行， 这是我近期的职业目标。为这一过渡做准备的初始阶段非常有希望。我 最近参加了宾夕法尼亚大学普林斯顿分校终身教职的面试 大学和西奈山医学院。Mt.Mt.西奈最近邀请我第二次访问，我 我正在等待宾夕法尼亚大学和普林斯顿大学的回复。为了帮助促进这一过渡，我将拥有协作性和 在造血(大卫·斯卡登博士)、急性髓细胞白血病(金伯利·斯泰格迈尔博士)和 LncRNA/计算生物学(约翰·里恩博士)。这些科学家致力于引导我通过 过渡过程，并帮助我在尽可能强大的位置上开始我的独立职业生涯。 一旦独立，我的研究将集中在AML细胞分化的表观遗传调控上。然而，我的 长期的愿景是将针对血统癌症的细胞命运决定的概念带到选定的 感兴趣的实体肿瘤。“分化疗法”在实体肿瘤中的研究还不够深入，但 干细胞和去分化程序显然与某些恶性肿瘤有关，如脑 癌症。因此，在拟议的资助期结束时，我的目标是建立以差异化为基础的 选定的脑癌筛查方法，并将致力于表征分子机制 使用遗传、生化、表观基因组学和动物模型的组合的屏幕热门。我相信这一点 指导将导致概念和治疗方面的进步，并将具有直接的临床意义。

Project Summary/Abstract I received my undergraduate degree from Cornell University, my PhD in molecular biology from Princeton University, and am currently a postdoctoral researcher and Special Fellow of the Leukemia and Lymphoma Society at Harvard Medical School and Boston Children's Hospital. After studying the molecular basis of breast cancer metastasis in graduate school, I transitioned to the field of epigenetics in the laboratory of Yang Shi for my postdoc. I have received extensive experimental and scholarly training in my postdoc. I learned the rigors of basic chromatin biochemistry in my first postdoctoral project, and recently published a co-first author paper based on this work. This study was featured as the cover article in the journal Cell and reported the identification of a novel metazoan epigenetic DNA modification, N6-methyladenine (6mA). My scholarly training has involved extensive scientific writing and has enabled me to write three successful postdoc fellowship applications (from the NIH, ACS, and LLS), two successful grants for Dr. Shi based on my work (from the Samuel Waxman Cancer Research Foundation and the Harvard Epigenetics Initiative), and one recently published review paper as a co-author in the journal Nature Reviews Molecular Biology. My current postdoctoral research merges my background in cancer biology with my recent training in chromatin biochemistry and focuses on the epigenetic basis of the differentiation block that characterizes acute myeloid leukemia (AML). AML is the most lethal hematological malignancy and is the cause of more than 10,000 annual US death. AML is typically treated by chemotherapy, though patients often relapse and have limited therapeutic options. The promyelocytic subtype of AML can be cured by “differentiation therapy” – induction of differentiation and inhibition of proliferation with retinoic acid – but this approach has been ineffective in other AML subtypes. Recent work has suggested that this block to non-APL AML differentiation is epigenetic in nature. Stable – yet reversible – chromatin alterations are thought to disable myeloid differentiation gene expression programs in these cells. I have recently established a robust genetic screening approach to uncover epigenetic regulators of non-APL AML differentiation programs. These screens have identified the Chaf1b subunit of the Caf-1 histone remodeling complex as a critical regulator of the Hoxa9- driven AML differentiation block. The overall goal of the work proposed in this application is to determine the molecular, chromatin-based mechanisms by which Chaf1b functions to regulate AML cell differentiation. The goal of my first Aim in this proposal is to use biochemical approaches to identify the proteins and long non- coding RNAs (lncRNAs) that associate with Chaf1b and the greater Caf-1 complex in AML cells. I will then focus on determining which of these interacting proteins and RNAs are most critical in recruiting Chaf1b to its target genomic loci and help sustain its activity as a differentiation-blocking protein. In Aim 2 I will use extensive epigenomic profiling approaches to identify the chromatin remodeling and transcriptomic processes that are altered as cells differentiate in response to inhibition of Chaf1b. I will then compare these epigenomic data to those obtained from my recent profiling of the normal, physiological myeloid differentiation program to determine whether loss of Chaf1b induces differentiation through typical or atypical mechanisms. Finally, in Aim 3 I will determine the potential of Chaf1b as a candidate therapeutic target by testing whether its inhibition induces differentiation and reduces disease burden or affects normal hematopoiesis in mouse AML models. While I am currently a postdoc, the proposed research is to be carried out as an independent investigator, which is my immediate career goal. The initial phase of preparing for this transition has been very promising. I have recently had interviews for tenure-track faculty positions at the University of Pennsylvania, Princeton University, and the Mount Sinai School of Medicine. Mt. Sinai has recently invited me for a second visit and I am awaiting responses from UPenn and Princeton. To help facilitate this transition, I will have collaborative and consultative support of field leaders in hematopoiesis (Dr. David Scadden), AML (Dr. Kimberly Stegmaier), and lncRNA/computational biology (Dr. John Rinn). These scientists are committed to guiding me through the transition process and helping me start my independent career in the strongest possible position. Once independent, my research will focus on epigenetic regulation of AML cell differentiation. However, my long term vision is to bring the concept of targeting cell fate decisions from blood lineage cancers to selected solid tumors of interest. “Differentiation therapy” has been underexplored in solid tumors, but the importance of stemness and de-differentiation programs has been clearly implicated in certain malignancies such as brain cancer. Therefore, toward the end of the proposed funding period, I will aim to establish differentiation-based screening methodology for selected brain cancers and will work on characterizing the molecular mechanisms of screen hits using a combination of genetic, biochemical, epigenomic, and animal models. I believe that this direction will lead to conceptual and therapeutic advances and will have direct clinical relevance.