H3K79 Methylation in Hematopoietic Stem Cell Development and MLL-Rearranged Leuk

描述（由申请人提供）：我提出了一个为期5年的职业发展计划，旨在了解表观遗传变化在正常造血发育和MLL重排白血病中的作用，同时建立造血干细胞移植的学术生涯。我将在分子生物学和临床血液学/肿瘤学的坚实基础上，在Stuart Orkin博士和Scott Armstrong博士的领导下，发展表观遗传调控，造血干细胞和白血病生物学方面的专业知识。 Orkin博士是造血发育和全基因组遗传学、表观遗传学和蛋白质组学研究的先驱和世界领导者。阿姆斯特朗博士是MLL重排白血病基因组方法和表观遗传变化的世界领导者。近年来，表观遗传修饰已开始被认为是一个动态和精细调控的过程，参与转录控制和主要的细胞命运决定。组蛋白甲基转移酶Dot 1 l可使组蛋白3的赖氨酸79（H3 K79）甲基化，最近被认为是混合谱系白血病（MLL）基因重排白血病的重要组成部分。未发表的数据也指出了在早期造血发育中的基本作用。我们已经为Dot 1 l产生了一个条件性功能丧失的小鼠模型，我们将使用这个模型来直接解决Dot 1 l在造血和白血病中的作用。 在具体目标1中，我们将表征造血发育和稳态成人造血过程中Dot 1 l失活的造血表型。我们将通过一系列的遗传和移植实验，评估胚胎造血发育，干细胞和祖细胞频率，自我更新和分化的体外和体内。我们还将研究H3 K79甲基化的丧失如何在全基因组水平上影响造血干细胞中的其他染色质修饰。 在具体目标2中，我们将检验以下假设：通过MLL融合蛋白错误募集DOT 1 L是MLL介导的白血病发生的基本步骤，并且是白血病细胞存活所需的。为此，我们将评估在我们的条件性Dot 11敲除小鼠模型中由常见融合M11-Af 4和M11-Af 9诱导的白血病的发展和维持。我们将在人MLL重排白血病细胞中进行DOT 1 L sh-RNA敲除实验。此外，我们计划与Stuart Schreiber博士合作，利用我们的模型来验证DOT 1 L的潜在小分子抑制剂。这些目标的实现将阐明良性和恶性造血中染色质调控的基本机制。 公共卫生相关性： 本文提出的研究计划将为造血干细胞的表观遗传调控以及良性和恶性造血发育提供有价值的见解。造血干细胞（HSC）是一种稀缺资源，了解控制HSC自我更新和扩增的遗传和表观遗传原则是血液学和干细胞移植领域的关键问题。此外，抑制白血病相关的表观遗传学变化可能显示出巨大的治疗希望的MLL重排白血病。

DESCRIPTION (provided by applicant): I present a 5-year career development plan that seeks to understand the role of epigenetic changes in normal hematopoietic development and MLL-rearranged leukemia, while establishing an academic career in Hematopoietic Stem Cell Transplantation. I will build on my strong foundation in molecular biology and clinical hematology/oncology to develop expertise in epigenetic regulation, hematopoietic stem cell, and leukemia biology under the leadership of Dr. Stuart Orkin and Dr. Scott Armstrong. Dr Orkin is a pioneer and world leader in hematopoietic development and genome wide genetic, epigenetic and proteomic studies. Dr Armstrong is a world leader in genomic approaches and epigenetic changes in MLL rearranged leukemias. In the recent years, epigenetic modifications have started to be recognized as a dynamic and finely regulated processe involved in transcriptional control and major cell fate decisions. The histone methyl transferase Dot1l, which methylates histone 3 at lysine 79 (H3K79), has recently been implicated as a crucial component in mixed lineage leukemia (MLL) gene rearranged leukemias. Unpublished data also point to a fundamental role in early hematopoietic development. We have generated a conditional loss of function mouse model for Dot1l, and we will use this model to directly address the role of Dot1l in hematopoiesis and leukemia. In specific aim 1 we will characterize the hematopoietic phenotype of Dot1l inactivation during hematopoietic development and steady state adult hematopoiesis. We will assess embyonic hematopoietic development, stem and progenitor cell frequencies, self-renewal and differentiation in vitro and in vivo through a series of genetic and transplantation experiments. We will also investigate how loss of H3K79 methylation affects other chromatin modifications in hematopoietic stem cells on a genome wide level. In specific aim 2 we will test the hypothesis that mis-recruitment of DOT1L through MLL-fusion proteins is a fundamental step in MLL-mediated leukemogenesis, and required for leukemia cell survival. To this end, we will assess the development and maintenance of leukemia induced by the common fusions Mll-Af4 and Mll-Af9 in our conditional Dot1l knockout mouse model. We will conduct DOT1L sh-RNA knock down experiments in human MLL-rearranged leukemia cell. In addition, we plan to utilize our models to validate potential small molecule inhibitors for DOT1L in collaboration with Dr. Stuart Schreiber. Execution of these aims will elucidate fundamental mechanisms of chromatin regulation in benign and malignant hematopoiesis. PUBLIC HEALTH RELEVANCE: The research plan presented here will provide valuable insight into epigenetic regulation of hematopoietic stem cells, and benign and malignant hematopoietic development. Hematopoietic stem cells (HSCs) are a scarce resource, and understanding the genetic and epigenetic principles that govern HSC self renewal and expansions are key questions in the field of hematology and stem cell transplantation. In addition, inhibition of leukemia-associated epigenetic changes may show great therapeutic promise for MLL-rearranged leukemias.