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

急性髓性白血病中 NUP98 重排和共同改变的遗传忠实模型

基本信息

批准号：
10443579
负责人：
Nicole Michmerhuizen
金额：
$ 6.76万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10443579
关键词：
Acute Erythroblastic Leukemia Acute Myelocytic Leukemia Affect Binding Binding Sites Biological Assay Bone Marrow Transplantation C-terminal CRISPR/Cas technology Chemoresistance Childhood Childhood Acute Myeloid Leukemia Chromatin Chromatin Remodeling Factor Chromatin Structure Chromosomal translocation Colony-forming units Complex DNA Binding DNA Sequence Alteration Data Set Development Developmental Gene Disease EP300 gene Engineering Epigenetic Process Erythroid Event FLT3 gene Funding Fusion Oncogene Proteins Future Gene Expression Gene Expression Profile Gene Expression Regulation Gene Fusion Genes Genetic Genetic Transcription Genomics Goals HOXA9 gene Hematopoietic Hematopoietic stem cells Histones Homeobox Genes Immunophenotyping In Vitro Individual Investigation Laboratories Lead Leukemic Cell Malignant Neoplasms Mediating Methods Modeling Molecular Monitor Monocytic leukemia Mus Mutation N-terminal Nuclear Export Nuclear Pore Complex Proteins Oncogenic Pathogenesis Pathogenicity Pathology Pathway interactions Patient-Focused Outcomes Patients Property Protein Export Pathway RB1 gene Receptor Protein-Tyrosine Kinases Recurrence Regulator Genes Research Research Proposals Resistance Role Sampling Scientist Specificity Survival Rate System Testing Therapeutic Time Training Transcription Coactivator Transcriptional Regulation Transposase Tumor Suppressor Proteins Validation WT1 gene Work base career childhood cancer mortality chimeric gene chromatin immunoprecipitation chromatin remodeling cofactor design effective therapy epigenetic regulation genomic locus high risk histone modification homeodomain human disease improved in vivo in vivo Model insight leukemia leukemogenesis mouse model multiple omics novel novel therapeutic intervention novel therapeutics nuclease overexpression patient population promoter self-renewal targeted treatment therapy resistant transcription factor transcriptome sequencing transcriptomics

项目摘要

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.

项目总结约5%的儿童中观察到涉及核孔蛋白98(NUP98)的染色体易位急性髓系白血病(AML)，与治疗抵抗和患者预后不良有关 (5年总存活率约为35%)。NUP98重排导致癌基因嵌合表达涉及NUP98 N-端区和30多个已鉴定的其中之一的C-端区的基因融合伴侣基因。伙伴基因通常具有具有关键功能特性的结构域，包括同源结构域部分(例如HOXA9)和在转录调控中的作用(例如NSD1，KDM5A)。在复合体中在基因调控所需的其他机制中，NUP98融合癌蛋白(FOS)与许多发育基因。这导致染色质结构发生变化，靶基因表达增加，和异常的造血自我更新。最近的白血病基因组测序研究，包括我的研究实验室的研究表明，NUP98融合通常伴随着不同的反复发生的遗传事件 AML的亚群，表明共改变在谱系特异性白血病发生中的重要性。这项研究该提案旨在阐明NUP98-重新排列的AML中共同改变的影响，从而建立在以前的基础上对遗传上忠诚的系统进行的研究可能会在这种高水平出现未知的治疗漏洞- 白血病风险亚群。目的1评估共改变基因在NUP98FO驱动的白血病发生中的作用。我将使用慢病毒过度表达和基于CRISPR/Cas9的方法在造血干细胞和祖细胞(HSPC)引入NUP98 FO和/或共改变，我会研究利用集落形成单位和骨髓检测这些变化的体内外后果分别进行移植试验。将使用免疫表型、病理学和基因组特征。以确定个体和组合遗传事件在不同疾病状态中的作用。Aim 2将检查当NUP98 FO和相关基因发生在转录和表观遗传水平上的分子机制共变可以单独表达，也可以协同表达。使用RNA测序，在靶标下切割并释放使用核酸酶(CUT&RUN)和转座酶可及染色质测序分析(ATAC-SEQ)，我将整合基因表达谱、DNA结合特性和组蛋白修饰，以及分别激活伴随着NUP98FO表达和/或共改变的染色质。这是一部综合性的作品该方法将为研究NUP98融合和基因调控效应提供可靠的数据集频繁的共变以及对其功能后果的验证。总而言之，这些研究将发现复发遗传事件与NUP98之间的协同作用对谱系特异性和分子水平的影响急性髓系白血病的重组，揭示了利用治疗来改善患者预后的新机会。