Characterization of novel subtypes in B progenitor acute lymphoblastic leukemia

B 祖细胞急性淋巴细胞白血病新亚型的特征

• 批准号: 10215447
• 负责人: Zhaohui Gu
• 金额: $ 24.9万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-13 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10215447
• 关键词: ATAC-seq; Acute Lymphocytic Leukemia; Adult; Adult Acute Lymphocytic Leukemia; Affect; Age; B-Lymphocytes; BCL2 gene; Binding Sites; Biological Assay; Biological Process; CRISPR/Cas technology; ChIP-seq; Childhood; Childhood Acute Lymphocytic Leukemia; Chromatin; Chromosomal Rearrangement; Classification; Clonality; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Coupled; DNA Sequence Alteration; Data; Data Analyses; Diagnostic; Ensure; Environment; Epidermal Growth Factor; Event; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Rearrangement; Genes; Genetic Transcription; Genomic approach; Genomics; Goals; Human; In Vitro; Knock-in; Knock-in Mouse; Lesion; Lymphoid; Malignant Childhood Neoplasm; Mentors; Missense Mutation; Modality; Modeling; Mus; Mutation; Oncogene Deregulation; Outcome; PAX5 gene; Pathway interactions; Patient-Focused Outcomes; Phase; Phosphotransferases; Pilot Projects; Play; Point Mutation; Protein Isoforms; RNA Interference; RNA analysis; Recurrence; Relapse; Residual state; Resources; Rest; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Saint Jude Children' s Research Hospital; Sample Size; Sampling; Structure; Subgroup; Taxonomy; Technology; Tertiary Protein Structure; Testing; Therapeutic; Transcript; Transposase; Untranslated RNA; Work; Xenograft procedure; analysis pipeline; base; chemotherapy; childhood cancer mortality; chimeric gene; differential expression; driver mutation; experience; experimental study; functional genomics; genetic signature; genome sequencing; genomic data; improved; individualized medicine; innovation; knock-down; leukemia; leukemogenesis; mouse model; new therapeutic target; novel; overexpression; progenitor; prognostic; role model; screening; single cell sequencing; skills; targeted treatment; therapeutic target; tool; transcription factor; transcriptome sequencing; whole genome

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.

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 RNA测序（RNA-SEQ）技术的进步，已经确定了许多复发性嵌合基因 导致了B-All和量身定制疗法的精制分类。不过，大约10-30％的B-所有情况不可能 分类为已建立的亚型，这些亚型被称为“ B-蛋白”，因此，一般化学疗法将是 应用和许多人的结果很差。这项研究将采用综合基因组数据分析来识别 新颖的B-ALL亚型，重点是B-其他情况。凭借先前工作的经验和技能，我将分析 来自2000年以上儿童和成人的RNA-seq数据所有病例，并根据不同的基因定义新型亚型 表达谱并共享遗传改变。病例缺乏RNA-Seq的驾驶员病变将受到 到整个基因组测序（WGS）以识别各种遗传改变。其余的未分类案件 随着非编码区域的遗传改变，将通过功能性基因组数据进行研究（芯片和ATAC- SEQ）提供机械注释。此外，将进行功能实验以探索 新确定的亚型定义的遗传改变的作用。在试点研究中，我分析了1,988个RNA- SEQ样品并定义了23个不同的B-all亚型，并确定了8个新颖的亚型。除了由 基因重排，我还观察到关键转录因子上的点突变可能在 定义新型亚型，其中包括PAX5 P80R（n = 44）和IKZF1 N159Y（n = 8）。在此提案中，我将扩展 样本量并用WGS询问其余的B-其他病例，以定义残留的新型亚型。通过 这项研究，我将提供明确的B-所有亚型，并最大程度地发出从B-other定义新的亚型的潜力 案例。作为单点突变定义亚型的示例，PAX5 P80R将在此彻底研究 提案。特别是，我将使用pax5加上其他密钥激活/抑制染色质标记。 研究PAX5 P80R特异性结合位点，再加上ATAC-SEQ的染色质可访问性信息。 使用CRISPR/CAS9敲入PAX5 P80R鼠标模型，我将使用Proleukemic的单细胞测序 白血病B细胞阐明了遗传改变与细胞水平上失调的基因之间的相关性。 此外，明显表达过度表达的基因MEGF10（多个表皮生长因子样域蛋白 10）在PAX5中，将通过体外和离体模型探索PAX5 P80R组以测试其在细胞定位中的作用 和白血病。通过RNAi或CRISPR的MEGF10的敲击或-out将应用于人P80R 异种移植物测试MEGF10是否可能是量身定制治疗的潜在目标。该提议的修改阶段 将在Charles Mullighan博士的领导下发生在圣裘德儿童研究医院 表征新颖的B-ALL亚型。独立阶段将集中于PAX5 P80R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 在我的提案中概述了我的成功过渡到独立性。