Translating genomic discoveries to improved outcomes for high risk acute leukemia

摘要 急性淋巴细胞白血病(ALL)是儿童癌症死亡的主要原因。我的研究目标是 使用整合的基因组和表观基因组图谱来定义驱动 白血病的发生和治疗都失败了，并利用这些信息来开发小鼠模型来翻译 这些发现带来了创新的治疗方法。我的研究项目已经修改了分子 ALL的分类学，已确定的定义ALL亚型的基因突变星座，已经剖析了 克隆进化的遗传学基础，确定了治疗干预的新靶点，特别是酪氨酸激酶 抑制剂，并建立了几个新的工程模型的高危B-祖细胞ALL。近期 进展包括在ALL复发时鉴定表观遗传调节因子的高频率突变，以及 证明特定的基因组改变扰乱了白血病细胞与 微环境，导致对治疗的抵触。这项研究计划将决定 基因损伤在诊断时出现或复发时丰富的基础，决定了对治疗的抵抗力， 并利用这些进行治疗干预。研究目标是(1)确定基因组的星座 以及表观基因组的改变，表征了整个年龄段的ALL的每个亚型，并识别 那些导致治疗失败的改变。这涉及到基因组和转录组测序 儿童和成人ALL，整合了全基因组、全基因组亚硫酸盐、转录组和染色质 对100例ALL病例进行标记测序，包括在诊断和复发时获得的匹配样本 以及相应的异种移植物。这对于识别导致疾病的所有编码和非编码突变至关重要， 系统研究遗传改变对染色质重塑的影响，并指导 开发和解释所有的小鼠模型。(2)执行多路功能丧失屏幕 利用建立癌基因融合的表达，结合RNA干扰和基因组编辑来解剖 白血病发生中多基因改变的相互作用。(3)使用基因特异性和功能丧失 筛查表观基因组改变在所有复发中的作用。这些包括详细的特征描述 针对700多个染色质修饰物的RNAi/CRISPR/Cas9筛选 B-ALL白血病模型中的基因。丰富的HITS及其对染色质建模和转录的影响 监管将与来自人类白血病细胞的数据进行比较；由此产生的模型将用于测试效果 表观遗传修饰剂在调节耐药性方面的研究。(4)用B-ALL小鼠模型进行解剖 细胞错位和“劫持”骨髓龛的作用以及这一现象在 抗药性。总之，这些方法提供了一种全面的策略来充分定义基因组 所有导致治疗失败的改变，并在逻辑实验中从机械上验证这些 指导进一步药物开发方法的系统。

ABSTRACT Acute lymphoblastic leukemia (ALL) is a leading cause of cancer death in children. The goal of my research is to use integrated genomic and epigenomic profiling to define the genomic alterations that drive leukemogenesis and treatment failure in ALL, and to use this information to develop mouse models to translate these discoveries to innovative therapeutic approaches. My research program has revised the molecular taxonomy of ALL, identified constellations of genetic mutations that define subtypes of ALL, has dissected the genetic basis of clonal evolution, identified new targets for therapeutic intervention, notably with tyrosine kinase inhibitors, and has established several new engineered models of high-risk B-progenitor ALL. Recent advances include identification of a high frequency of mutations in epigenetic regulators at relapse in ALL, and demonstrating that specific genomic alterations perturb the interaction of leukemic cells with the microenvironment, resulting in resistance to therapy. This research proposal will determine the mechanistic basis by which genetic lesions present at diagnosis, or enriched at relapse, determine resistance to therapy, and exploit these for therapeutic intervention. Research goals are (1) to identify the constellations of genomic and epigenomic alterations that characterize each subtype of ALL across the age spectrum, and identifying those alterations that cause treatment failure. This involves genome and transcriptome sequencing of childhood and adult ALL, and integrated whole genome, whole genome bisulfite, transcriptome and chromatin mark sequencing of a cohort of 100 ALL cases, including matched samples obtained at diagnosis and relapse and corresponding xenografts. This is essential to identify all coding and non-coding mutations driving disease, to systematically examine the effect of genetic alterations on chromatin remodeling, and to guide the development and interpretation of mouse models of ALL. (2) To perform multiplexed loss-of-function screens using expression of founding oncogenic fusions, coupled with RNA interference and genome editing to dissect the interaction of polygenic alterations in leukemogenesis. (3) To use gene-specific and loss-of-function screens to examine the role of epigenomic alterations in ALL relapse. These include detailed characterization of Crebbp knockin models of ALL, and RNAi/CRISPR/Cas9 screens targeting over 700 chromatin modifier genes in B-ALL leukemia models. Enriched hits, and their effects on chromatin modeling and transcriptional regulation will be compared to data from human leukemic cells; and the resulting models used to test the effect of epigenetic modifying agents on modulating drug resistance. (4) To use mouse models of B-ALL to dissect the role of cellular mislocalization and “hijacking” of the bone marrow niche and the role of this phenomenon in drug resistance. Together, these approaches provide a comprehensive strategy to fully define the genomic alterations driving treatment failure in ALL, and to mechanistically validate these in logical experimental systems to guide further drug development approaches.