Genetically faithful modeling of NUP98 rearrangement and co-alterations in acute myeloid leukemia

项目摘要 在大约5%的儿童中观察到涉及核孔蛋白98（NUP 98）的染色体易位。 急性髓性白血病（AML），并与耐药性和患者预后不良相关 （约35%的5年总生存率）。NUP 98重排导致致癌嵌合基因的表达 涉及NUP 98的N-末端区域和30多个鉴定的 伴侣基因伴侣基因通常具有关键功能特性的结构域，包括 同源结构域部分（例如HOXA 9）和在转录调节中的作用（例如NSD 1、KDM 5A）。在复杂 NUP 98融合癌蛋白（FO）与基因调控所需的其他机制结合， 许多发育基因。这导致染色质结构的变化，靶基因表达的增加， 和异常的造血自我更新最近的白血病基因组测序研究，包括我的 实验室，已经表明NUP 98融合通常伴随着在不同的细胞中重复发生的遗传事件， AML的亚群，提示共改变在谱系特异性白血病发生中的重要性。本研究 一项提案旨在阐明NUP 98重排AML中共同改变的影响，从而建立在以前的基础上， 对基因忠实系统的研究，可能会在这种高- 白血病风险亚群。目的1评价共改变基因在NUP 98 FO驱动的白血病发生中的作用。 我将使用慢病毒过表达和基于CRISPR/Cas9的方法来进行基因编辑， 造血干细胞和祖细胞（HSPC）引入NUP 98 FO和/或共改变，我将研究 使用集落形成单位和骨髓观察这些改变的体外和体内结果 移植试验，分别。将使用免疫表型、病理学和基因组表征 以确定个体和组合遗传事件对不同疾病状态的作用。目标2将检查 当NUP 98 FO和相关的转录和表观遗传水平发生的分子机制， 共同改变是单独表达的或一致表达的。使用RNA测序、靶下切割和释放 使用核酸酶（CUT&RUN），和使用测序（ATAC-seq）的转座酶可降解染色质测定， 我将整合基因表达谱，DNA结合特性和组蛋白修饰，以及 分别激活伴随NUP 98 FO表达和/或共改变的染色质。这种多基因的 该方法将为NUP 98融合的基因调控作用的研究提供一个强大的数据集， 频繁的共同改变以及验证其功能后果。这些研究将 揭示重复遗传事件和NUP 98之间合作的谱系特异性和分子影响 AML中的基因重排，揭示了治疗开发以改善患者结局的新机会。

PROJECT SUMMARY Chromosomal translocations involving Nucleoporin 98 (NUP98) are observed in approximately 5% of pediatric acute myeloid leukemia (AML) and are associated with resistance to therapy and poor patient outcomes (approximately 35% 5 year overall survival). NUP98 rearrangements lead to expression of oncogenic chimeric gene fusions involving the N-terminal region of NUP98 and the C-terminal region of one of over 30 identified partner genes. The partner genes commonly have domains with key functional properties, including homeodomain moieties (e.g. HOXA9) and roles in transcriptional regulation (e.g. NSD1, KDM5A). In complex with other machinery needed for gene regulation, NUP98 fusion oncoproteins (FOs) bind to the promoters of many developmental genes. This leads to changes in chromatin structure, increased expression of target genes, and aberrant hematopoietic self-renewal. Recent leukemia genomic sequencing studies, including those by my laboratory, have shown that NUP98 fusions are commonly accompanied by recurring genetic events in distinct subsets of AML, suggesting the importance of co-alterations in lineage-specific leukemogenesis. This research proposal seeks to elucidate the effects of co-alterations in NUP98-rearranged AML, thus building on previous studies with genetically faithful systems that could present undiscovered therapeutic vulnerabilities in this high- risk leukemia subset. Aim 1 will evaluate the effects of co-altered genes in NUP98 FO-driven leukemogenesis. I will use lentiviral overexpression and CRISPR/Cas9-based approaches to perform gene editing in hematopoietic stem and progenitor cells (HSPCs) to introduce NUP98 FO and/or co-alteration, and I will study the in vitro and in vivo consequences of these alterations using colony forming unit and bone marrow transplantation assays, respectively. Immunophenotyping, pathology, and genomic characterization will be used to determine the role of individual and combined genetic events on distinct disease states. Aim 2 will examine the molecular mechanisms that occur at the transcriptional and epigenetic level when NUP98 FO and relevant co-alterations are expressed alone or in concert. Using RNA sequencing, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), I will integrate the gene expression profiles, DNA binding properties and histone modifications, and regions of activate chromatin, respectively, that accompany expression of NUP98 FO and/or co-alteration. This multiomic approach will provide a robust dataset for the investigation of the gene-regulatory effects of NUP98 fusions and frequent co-alterations as well as for validation of their functional consequences. Together, these studies will uncover lineage-specific and molecular impacts of cooperation between recurrent genetic events and NUP98 rearrangements in AML, revealing novel opportunities for therapeutic exploitation to improve patient outcomes.