Characterization of novel subtypes in B progenitor acute lymphoblastic leukemia

项目摘要 B祖细胞急性淋巴细胞白血病（B-ALL）仍然是儿童癌症死亡的主要原因。与 随着RNA测序（RNA-seq）技术的发展，已经鉴定了许多重复的嵌合基因， 导致B-ALL的精确分类和定制治疗。尽管如此，大约10-30%的B-ALL病例不能被治疗。 分为已确定的亚型，称为“B-其他”，因此一般化疗将 结果，许多人的结果很差。本研究将应用整合基因组数据分析， 新型B-ALL亚型，重点关注B-other病例。根据以往工作的经验和技巧，我将分析 来自2000多例儿童和成人ALL病例的RNA-seq数据，并根据不同的基因定义新的亚型 表达谱和共有的遗传改变。来自RNA-seq的缺乏驱动病变的病例将接受 全基因组测序（WGS），以确定各种遗传变异。剩下的未分类案件 将通过功能基因组数据（ChIP和ATAC- seq）以提供机械注释。此外，还将进行功能实验，以探索 新发现的亚型定义遗传改变的作用。在初步研究中，我分析了1,988个RNA- seq样本，并确定了23个不同的B-ALL亚型，其中8个新的确定。除了定义为 基因重排，我还观察到关键转录因子上的点突变可能在 定义了新的亚型，包括PAX 5 P80 R（n=44）和IKZF 1 N159 Y（n=8）。在这份提案中，我将扩大 其余B-其他病例用WGS进行询问，确定剩余的新亚型。通过 在这项研究中，我将提供明确的B-ALL亚型，并最大限度地从B-其他亚型中定义新的亚型。 例作为单点突变定义的亚型的范例，PAX 5 P80 R将在本研究中被深入研究。 提议具体来说，我将通过ChIP-seq使用PAX 5加上其他关键的激活/抑制染色质标记， 研究PAX 5 P80 R特异性结合位点，结合来自ATAC-seq的染色质可及性信息。 在CRISPR/Cas9基因敲入Pax 5 P80 R小鼠模型中，我将使用白血病前期和白血病晚期的单细胞测序。 白血病B细胞，以阐明细胞水平上遗传改变和失调基因之间的相关性。 此外，显著过表达的基因MEGF 10（多表皮生长因子样结构域蛋白）， 10)在PAX 5中，将通过体外和离体模型探索P80 R组，以测试其在细胞定位中的作用 和白血病发生。通过RNAi或CRISPR敲低或敲除MEGF 10将应用于人P80 R 异种移植来测试MEGF 10是否可以成为定制治疗的潜在靶点。本提案的辅导阶段 将发生在圣裘德儿童研究医院，在查尔斯Mullighan博士，并将完成的目标， 表征新型B-ALL亚型。独立阶段将专注于PAX 5 P80 R的功能研究 或其他研究不足的亚型。机构资源和学术环境以及计划的课程 我的建议中所概述的将确保我成功地过渡到独立。

Project Summary B progenitor acute lymphoblastic leukemia (B-ALL) remains a leading cause of childhood cancer death. With the advances in RNA sequencing (RNA-seq) technology, many recurrent chimeric genes have been identified that has led to refined classification of B-ALL and tailored therapies. Still, around 10-30% B-ALL cases could not be classified into the established subtypes, which are termed as “B-other”, thus general chemotherapy will be applied and the outcome for many is poor. This study will apply integrative genomic data analysis to identify novel B-ALL subtypes with a focus on B-other cases. With the experience and skills from prior work, I will analyze RNA-seq data from over 2000 childhood and adult ALL cases and define novel subtypes based on distinct gene expression profiles and shared genetic alterations. Case lacking driver lesions from RNA-seq will be subjected to whole genome sequencing (WGS) to identify various genetic alterations. The remaining unclassified cases with the genetic alterations in non-coding regions will be studied by functional genomic data (ChIP and ATAC- seq) to provide mechanistic annotation. Furthermore, functional experiments will be performed to explore the role of the newly identified subtype-defining genetic alterations. In the pilot study, I have analyzed 1,988 RNA- seq samples and defined 23 distinct B-ALL subtypes, with 8 novel ones identified. Besides the ones defined by gene rearrangements, I also observed point mutations on key transcription factors could play potent role in defining novel subtypes, which include PAX5 P80R (n=44) and IKZF1 N159Y (n=8). In this proposal, I will expand the sample size and interrogate the rest B-other cases with WGS to define the residual novel subtypes. Through this study, I will provide definitive B-ALL subtypes and maximize the potential of defining new ones from B-other cases. As an exemplar of single-point-mutation-defined subtype, PAX5 P80R will be thoroughly studied in this proposal. Specifically, I will use PAX5 plus other key activating/repressing chromatin marks through ChIP-seq to study PAX5 P80R specific binding sites, coupled with the chromatin accessibility information from ATAC-seq. With the CRISPR/Cas9 knock-in Pax5 P80R mouse model, I will use single-cell sequencing of preleukemic and leukemic B cells to elucidate the correlation between genetic alterations and deregulated genes on cellular level. Moreover, the markedly overexpressed gene MEGF10 (Multiple Epidermal Growth Factor-Like Domains Protein 10) in PAX5 P80R group will be explored through in vitro and ex vivo models to test its role in cellular localization and leukemogenesis. Knock-down or -out of MEGF10 through RNAi or CRISPR will be applied in human P80R xenografts to test if MEGF10 could be a potential target for tailored therapy. The mentored phase of this proposal will occur at St. Jude Children’s Research Hospital, under Dr. Charles Mullighan, and will finish the aim of characterizing novel B-ALL subtypes. The independent phase will focus on the functional studies of PAX5 P80R or other under-studied subtypes. The institutional resources and academic environment and the planned courses outlined in my proposal will ensure my successful transition to independence.