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

项目概要/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 大学，现为白血病和淋巴瘤研究室博士后研究员和特约研究员 哈佛医学院和波士顿儿童医院协会。研究了其分子基础后 研究生阶段乳腺癌转移，我在杨实验室转入表观遗传学领域 施为我的博士后。我在博士后期间接受了广泛的实验和学术培训。我学到了 我的第一个博士后项目中基本染色质生物化学的严格性，最近发表了共同第一作者 基于这项工作的论文。这项研究作为封面文章发表在《细胞》杂志上，并报道了 鉴定出一种新型后生动物表观遗传 DNA 修饰 N6-甲基腺嘌呤 (6mA)。我的学术 培训涉及广泛的科学写作，使我能够撰写三篇成功的博士后文章 奖学金申请（来自 NIH、ACS 和 LLS），基于我的工作为石博士成功提供了两项资助 （来自塞缪尔·韦克斯曼癌症研究基金会和哈佛表观遗传学计划），以及一位 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的培训结合起来 染色质生物化学，重点关注表征急性的分化块的表观遗传基础 骨髓性白血病（AML）。 AML 是最致命的血液恶性肿瘤，导致超过 美国每年有一万人死亡。 AML 通常通过化疗治疗，但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“分化治疗”治愈—— 用视黄酸诱导分化并抑制增殖——但这种方法已被 对其他 AML 亚型无效。最近的工作表明，这种阻断非 APL AML 的分化 本质上是表观遗传。稳定但可逆的染色质改变被认为会导致骨髓细胞功能丧失 这些细胞中的分化基因表达程序。我最近建立了强大的基因筛查 揭示非 APL AML 分化程序的表观遗传调节因子的方法。这些屏幕有 确定 Caf-1 组蛋白重塑复合物的 Chaf1b 亚基是 Hoxa9- 的关键调节因子 驱动的 AML 分化块。本申请中提出的工作的总体目标是确定 Chaf1b 通过基于染色质的分子机制来调节 AML 细胞分化。 我在这个提案中的第一个目标是使用生化方法来识别蛋白质和长非 编码 RNA (lncRNA)，与 AML 细胞中的 Chaf1b 和更大的 Caf-1 复合体相关。然后我会 重点确定这些相互作用的蛋白质和 RNA 中哪些对于招募 Chaf1b 至其自身至关重要 靶向基因组位点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 广泛的表观基因组分析方法来识别染色质重塑和转录组过程 当细胞响应 Chaf1b 的抑制而分化时，这些变化就会发生改变。然后我将比较这些表观基因组 数据来自我最近对正常生理性骨髓分化程序的分析 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 目标 3 我将通过测试 Chaf1b 是否受到抑制来确定 Chaf1b 作为候选治疗靶点的潜力 在小鼠 AML 模型中诱导分化并减轻疾病负担或影响正常造血功能。 虽然我目前是博士后，但拟议的研究将以独立研究者的身份进行， 这是我近期的职业目标。为这一转变做准备的初始阶段非常有希望。我 最近接受了宾夕法尼亚大学普林斯顿大学终身教授职位的面试 大学和西奈山医学院。西奈山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。为了帮助促进这一转变，我将进行协作和 造血领域领导者（David Scadden 博士）、AML（Kimberly Stegmaier 博士）和 lncRNA/计算生物学（John Rinn 博士）。这些科学家致力于指导我完成 过渡过程并帮助我以最有利的位置开始我的独立职业生涯。 独立后，我的研究将集中于 AML 细胞分化的表观遗传调控。然而，我的 长期愿景是将针对血系癌症的细胞命运决定的概念引入选定的细胞 感兴趣的实体瘤。 “分化疗法”在实体瘤中尚未得到充分探索，但其重要性 干细胞和去分化程序明显与某些恶性肿瘤有关，例如脑部恶性肿瘤 癌症。因此，在拟议的资助期结束时，我的目标是建立基于差异化的 选定脑癌的筛查方法，并将致力于表征分子机制 使用遗传、生化、表观基因组和动物模型的组合来提高屏幕点击率。我相信这 方向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.