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 RNA测序(RNA-seq)技术的进展，已发现许多重复的嵌合基因 导致了对B-ALL的精细分类和量身定制的治疗。尽管如此，大约10%-30%的B-所有病例都不可能 归类为已建立的亚型，称为“B-其他”，因此全身化疗将 但对许多人来说，结果并不理想。这项研究将应用综合基因组数据分析来识别 新奇的B-所有亚型，重点是B-其他病例。凭借之前工作中的经验和技能，我将分析 来自2000多名儿童和成人ALL病例的RNA-SEQ数据，并根据不同的基因定义新的亚型 表达特征和共同的基因改变。缺少rna-seq的驾驶员皮损的病例将接受 到全基因组测序(WGS)以识别各种基因改变。其余的非机密案件 非编码区的遗传变化将通过功能基因组数据进行研究(CHIP和ATAC- SEQ)来提供机械性的注释。此外，还将进行功能性实验，以探索 新发现的亚型定义基因改变的作用。在初步研究中，我分析了1,988个RNA- SEQ样本，定义了23个不同的B-ALL亚型，其中8个新亚型被鉴定。除了由定义的 基因重排，我还观察到关键转录因子的点突变可能在 定义了新的亚型，包括PAX5 P80R(n=44)和IKZF1 N159Y(n=8)。在这个提案中，我将扩展 样本大小和询问其余B-其他病例与WGS，以确定剩余的新亚型。穿过 这项研究，我将提供明确的B-ALL亚型，并最大限度地从B-OTHER定义新的亚型 案子。作为单点突变亚型的样本，PAX5 P80R将在本论文中得到深入研究 求婚。具体地说，我将使用PAX5加上其他关键的激活/抑制染色质标记通过芯片序列来 研究PAX5 P80R特异结合位点，结合来自ATAC-seq的染色质可及性信息。 对于CRISPR/Cas9敲入Pax5 P80R小鼠模型，我将使用白血病前期和 在细胞水平上阐明基因改变与基因失控之间的关系。 此外，显著过表达的基因MEGF10(多个表皮生长因子样域蛋白 10)在PAX5组中，P80R将通过体外和体外模型进行探索，以测试其在细胞定位中的作用 和白血病的发生。通过RNAi或CRISPR敲除或敲除MEGF10将应用于人P80R 以测试MEGF10是否可能成为量身定制治疗的潜在靶点。此建议书的指导阶段 将在圣犹大儿童研究医院举行，由查尔斯·穆莱兰博士领导，并将完成 表征新奇的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。院校资源、学术环境与课程设置 我的提案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.