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：
10599975
负责人：
Nicole Michmerhuizen
金额：
$ 2.6万
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 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10599975
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 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.

项目概要在大约 5% 的儿科患者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)，与治疗耐药和患者预后不良相关（5 年总生存率约为 35%）。 NUP98 重排导致致癌嵌合体的表达基因融合涉及 NUP98 的 N 末端区域和 30 多个已识别的其中之一的 C 末端区域伴侣基因。伴侣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）和转录调控中的作用（例如 NSD1、KDM5A）。在复杂的与基因调控所需的其他机制一样，NUP98 融合癌蛋白 (FO) 与以下基因的启动子结合：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，包括我的研究实验室表明，NUP98 融合通常伴随着不同的重复遗传事件 AML 的子集，表明共同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 重排 AML 中共同改变的影响，从而建立在以前的基础上对遗传忠实系统的研究可能会在这种高风险中呈现未被发现的治疗漏洞危险白血病亚群。目标 1 将评估共同改变的基因在 NUP98 FO 驱动的白血病发生中的影响。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NUP98 FO和/或共同改变，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后果分别进行移植测定。将使用免疫表型、病理学和基因组表征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 NUP98 FO 和相关的转录和表观遗传水平上发生的分子机制共同改变可以单独或协同表达。使用 RNA 测序、目标下的切割和释放使用核酸酶 (CUT&RUN) 和使用测序 (ATAC-seq) 检测转座酶可及的染色质，我将整合基因表达谱、DNA 结合特性和组蛋白修饰以及区域分别激活伴随 NUP98 FO 表达和/或共同改变的染色质。这个多组学该方法将为研究 NUP98 融合的基因调控效应提供强大的数据集频繁的共同改变以及验证其功能后果。这些研究共同将揭示反复发生的遗传事件与 NUP98 之间合作的谱系特异性和分子影响 AML 中的重排，揭示了治疗开发以改善患者预后的新机会。