The role of epigenetic heterogeneity in CLL evolution Admin supplement

慢性淋巴细胞白血病（CLL）目前是无法治愈的。尽管有有效的治疗，但这种疾病总是复发，部分原因是它的进化能力。我们已经表明，预处理白血病内部遗传异质性预示着克隆进化导致疾病复发。然而，细胞表型及其适合选择的结果是遗传和表观遗传的改变。因此，癌症进化研究的一个主要挑战是整合遗传和表观遗传异质性。在初步研究中，我们发现CLL的样本内表观遗传异质性增加。为了了解这种异质性的基础，我们研究了大约100个原发性CLL样本的大量平行亚硫酸盐测序中单个reads中邻近CpGs甲基化状态的一致性。我们证明，大多数异质性源于无序甲基化，随机表观遗传漂变的一种形式。紊乱的甲基化影响转录调控的重要区域，并与启动子甲基化和转录沉默之间的关系脱钩有关。最后，无序甲基化受到选择的影响，可能促进克隆进化。我假设紊乱的甲基化影响组蛋白修饰和转录，从而促进CLL的进化。为了确定无序甲基化的影响，我们提出了以下独立但相互关联的特定目标：(1)为了研究其与组蛋白修饰和转录的关系，我们将制作全面的组蛋白ChIP-seq制图和针对抑制性组蛋白标记的chip -亚硫酸盐-seq。我们将整合多维数据来推断表观遗传样本内异质性，并用单细胞RNAseq验证这一点，以评估细胞间的变异性作为甲基化障碍的功能。(2)我们将开发一种统计推断工具，在考虑背景随机变化的情况下，检测癌症中假定的甲基化“驱动”事件。(3)为了研究无序甲基化对克隆进化和临床结果的影响，我们将对350例接受统一治疗的患者的预处理样本和80例复发样本进行遗传和表观遗传异质性分析。目前还没有有效的治疗策略来抑制癌症的发展。因此，这些研究解决了尚未满足的治疗需求。最后，在这份申请中，我概述了一个5年的职业发展计划，以实现我的目标，成为一名转化癌症生物学的独立研究者，精通大数据科学方法论。我组建了一个由国际公认的专家组成的指导委员会，提供科学和职业指导。我将追求密集的教学课程和实践培训与领先的专家，建立一个强大的计算和统计基础。最后，丹娜-法伯癌症研究所是实现我的科学和职业目标的理想环境，因为它有出色的研究社区，强调大数据科学，以及培养独立医生科学家的良好记录。

DESCRIPTION: Chronic lymphocytic leukemia (CLL) is currently incurable. Despite effective treatments, the disease invariably recurs due, in part, to its ability to evolve. We have shown that pretreatment intra-leukemic genetic heterogeneity foreshadows clonal evolution leading to disease relapse. Nevertheless, the cellular phenotype and its fitness for selection result from both genetic and epigenetic alterations. Therefore, a major challenge in the study of cancer evolution is to integrate genetic and epigenetic heterogeneity. In preliminary studies, we found increased intra-sample epigenetic heterogeneity in CLL. To understand the basis of this heterogeneity, we studied the uniformity of the methylation status of neighboring CpGs contained within individual reads from massively parallel bisulfite sequencing of ~100 primary CLL samples. We demonstrated that most of the heterogeneity stems from disordered methylation, a form of stochastic epigenetic drift. Disordered methylation affected regions important to transcriptional regulation and was associated with a decoupling of the relationship between promoter methylation and transcriptional silencing. Finally, disordered methylation was subjected to selection and may facilitate clonal evolution. I hypothesize that disordered methylation impacts histone modification and transcription, thereby enhancing CLL evolution. To define the impact of disordered methylation, we propose the following independent yet interrelated Specific Aims: (1) To examine its relationship to histone modification and transcription, we will produce comprehensive histone ChIP-seq mapping and ChIP-bisulfite-seq directed at repressive histone marks. We will integrate the multidimensional data to infer epigenetic intra-sample heterogeneity and validate this with single-cell RNAseq to assess cell-to-cell variability as a function of methylation disorder. (2) We will develop a statistical inferece tool to detect putative methylation "driver" events in cancer taking into account background stochastic variation. (3) To study the impact of disordered methylation on clonal evolution and clinical outcome, we will integrate genetic and epigenetic heterogeneity analysis in pretreatment samples from 350 patients who received uniform treatment, and 80 relapse samples. There are no therapeutic strategies currently available to curb cancer evolution. Thus, these studies address an unmet therapeutic need. Finally, in this application, I have outlined a 5-year career development plan to meet my goal of becoming an independent investigator in translational cancer biology, proficient in big data science methodology. I have assembled a Mentorship Committee of internationally recognized experts to provide scientific and career mentorship. I will pursue intensive didactic coursework and hands-on training with leading experts, to develop a strong computational and statistical foundation. Finally, Dana-Farber Cancer Institute is the ideal environment for attaining my scientific and career goals, given its outstanding research community, emphasis on big data science, and an excellent track record of training independent physician-scientists.