The Role of CHAF1B in Maintaining Malignant Leukemia Stem Cells

项目摘要 患有唐氏综合征（DS）的儿童患急性白血病的风险很大，这表明 21号染色体上有白血病基因染色质组装因子1B（CHAF 1B）， 位于DS关键区，是异三聚体CAF 1染色质组装复合物的成员， 负责在S期期间在复制叉处沉积H3/H4异二聚体。CHAF 1B水平 在DS-AML患者样本以及非DS AML细胞系和原发性AML样本中升高， 与健康的样品相比。虽然在大多数肿瘤中，表达升高与预后不良相关， CHAF 1B促进白血病发生的机制尚不清楚。我的初步数据显示CHAF 1B 直接结合与髓样分化的启动子和增强子相关的染色质的离散区域 基因.髓系白血病细胞中Chaf 1b的缺失导致染色质上的CHAF 1B被替换为 包括CEBPA在内的促分化转录因子和随后的髓样细胞活化， 分化转录程序。因此，我建议CHAF 1B须维持 在MLL-AF 9白血病细胞的未分化状态下，通过作为转录调节因子的作用，在这份提案中，我 将研究CHAF 1B依赖性维持白血病干细胞的机制。核心假设是 CHAF 1B通过竞争染色质占据来维持骨髓白血病细胞的未分化状态 在分化基因的启动子和增强子处具有转录因子。这场比赛导致 这些基因的表达减少，并导致白血病原始细胞表型的维持。 在目标1中，我将探索CHAF 1B蛋白的不同结构域如何有助于 维持骨髓性白血病细胞的未分化状态。然后，在目标2中，我将CHAF 1B建立为 通过确定其对血液学进展的贡献，确定AML（包括DS-AML）的治疗靶点 体内肿瘤和体外原代人类患者样品。我的最终目标是利用获得的信息 在K99/R 00奖期间，开发可以阻断CHAF 1B功能的新型小分子化合物， 通过分化在体内控制AML肿瘤。我的职业目标是成为一名独立调查员和领导者 在染色质组装及其对造血系统恶性肿瘤的影响领域， 开发治疗白血病的新策略。在指导K99阶段，我的主要技术 目标是精通大规模NGS数据集和基本蛋白质组学的分析。的影响 K99阶段将通过我与Dana Farber癌症中心的Scott Armstrong博士的合作得到加强， 哈佛大学和韦恩州立大学的葛玉斌博士，允许我在外面扩展我的训练 我的家乡西北大学John Crispino博士和我的顾问团队将指导我 完成我的博士后培训，并帮助我过渡到独立。

PROJECT SUMMARY Children with Down syndrome (DS) are at substantial risk of developing acute leukemia, which suggests that there are leukemia promoting genes on chromosome 21. Chromatin Assembly Factor 1B (CHAF1B), which resides in the DS critical region, is a member of the heterotrimeric CAF1 chromatin assembly complex that is responsible for depositing H3/H4 heterodimers at the replication fork during S-phase. CHAF1B levels are elevated in DS-AML patient samples, as well as non-DS AML cell lines and primary AML samples when compared to healthy samples. While elevated expression is associated with poor prognosis in most tumors, the mechanism by which CHAF1B promotes leukemogenesis is unknown. My preliminary data show that CHAF1B directly binds discrete regions of chromatin associated with promoters and enhancers of myeloid differentiation genes. Deletion of Chaf1b in myeloid leukemia cells results in replacement of CHAF1B on the chromatin with the pro-differentiation transcription factors including CEBPA and the subsequent activation of a myeloid differentiation transcriptional program. Therefore, I propose that CHAF1B is required to maintain the undifferentiated state of MLL-AF9 leukemic cells through a role as a transcriptional regulator. In this proposal, I will study the mechanism of CHAF1B-dependent maintenance of leukemic stem cells. The central hypothesis is CHAF1B maintains the undifferentiated state of myeloid leukemic cells by competing for chromatin occupancy with transcription factors at the promoters and enhancers of differentiation genes. This competition leads to reduced expression of these genes and the resulting maintenance of the leukemic blast phenotype. In Aim 1, I will explore the mechanism of how the different domains of the CHAF1B protein contribute to maintaining the undifferentiated state of myeloid leukemic cells. Then, in Aim 2, I will establish CHAF1B as a therapeutic target in AML (including DS-AML) by determining its contribution to the progression of hematologic tumors in vivo and primary human patient samples in vitro. My ultimate goal is to use the information gained during the K99/R00 award to develop novel small molecule compounds that can block CHAF1B function and control AML tumors in vivo by differentiation. My career goal is to become an independent investigator and leader in the field of chromatin assembly and its effects on hematopoietic malignancies with the ultimate goal of developing new therapeutic strategies to treat leukemia. During the mentored K99 phase, my main technical goal is to become proficient at analysis of large-scale NGS data sets and basic proteomics. The impact of the K99 phase will be enhanced by my collaborations with Dr. Scott Armstrong at Dana Farber Cancer Center at Harvard University and Dr. Yubin Ge at Wayne State University, by allowing me to expand my training outside my home institution of Northwestern University. Dr. John Crispino and my advisory team will guide me through the completion of my postdoctoral training and assist me with my transition to independence.