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

• 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
• 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

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.

项目摘要 在大约5％的小儿中观察到涉及核孔98（NUP98）的染色体易位。 急性髓样白血病（AML），与对治疗的抗药性和患者预后不良有关 （大约35％5年的总生存率）。 NUP98重排导致致癌嵌合体的表达 涉及NUP98的N末端区域的基因融合和30多个确定的C末端区域 伴侣基因。伴侣基因通常具有具有关键功能属性的域，包括 同源域部分（例如HOXA9）和转录调节中的角色（例如NSD1，KDM5A）。复杂 使用基因调节所需的其他机械，NUP98融合癌蛋白（FOS）与启动子结合 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 实验室表明，NUP98融合通常伴随着不同的遗传事件 AML的子集，表明共同变化在谱系特异性白血病发生中的重要性。这项研究 提案旨在阐明NUP98重建AML的共同变量的影响，从而建立在先前 对遗传忠实的系统的研究，这些系统可能会在这种高度呈现这种高度的治疗脆弱性中 风险白血病子集。 AIM 1将评估共塑基因在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的方法进行基因编辑 造血茎和祖细胞（HSPC）引入NUP98 FO和/或共同化，我将研究 这些改变的体外和体内后果，使用菌落形成单元和骨髓 分别进行移植测定。将使用免疫表型，病理和基因组表征 确定个体和遗传事件在不同疾病状态的作用。 AIM 2将检查 NUP98 FO和相关的转录和表观遗传水平发生的分子机制 共同变化单独或共同表示。使用RNA测序，目标下的裂解并释放 使用核酸酶（切割和运行），并使用测序（ATAC-SEQ）（ATAC-SEQ）进行转座酶可访问的测定法测定 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和/或共同化表达的染色质。这个多媒体 方法将为调查NUP98融合的基因调节作用和 频繁的共同变化以及验证其功能后果。这些研究将在一起 发现复发遗传事件与NUP98之间合作的谱系特异性和分子影响 AML的重排，揭示了治疗性剥削的新机会，以改善患者预后。