Functional and Pharmacologic Investigation of the NUP98 Fusion Oncoprotein Interactome

NUP98 融合癌蛋白相互作用组的功能和药理学研究

• 批准号: 10724053
• 负责人: Nicole Michmerhuizen
• 金额: $ 14.1万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10724053
• 关键词: Acute Myelocytic Leukemia; Address; Binding; Bioinformatics; Biological Assay; Bone Marrow Transplantation; C-terminal; CRISPR screen; CRISPR/Cas technology; Cell Death; Cell Nucleus; Cell Survival; Cells; Childhood Acute Myeloid Leukemia; Childhood Leukemia; Chromatin; Chromatin Structure; Chromosomal Rearrangement; Chromosomal translocation; Colony-forming units; Complex; DNA Binding; Data; Data Set; Developmental Gene; Disadvantaged; Disease; Epigenetic Process; Fusion Oncogene Proteins; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Fusion; Genes; Goals; Guide RNA; HOXA9 gene; Hematopoietic; Hematopoietic stem cells; Histone Acetylation; Immunoprecipitation; In Vitro; Investigation; Lead; Libraries; Liquid substance; Lysine; MLL gene; Malignant - descriptor; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator; Membrane; Modeling; Mus; N-terminal; Nuclear Pore Complex Proteins; Oncogene Deregulation; Oncogenic; Organelles; Outcome; Patient-Focused Outcomes; Pharmacology; Phase; Phenotype; Property; Proteins; Reader; Recurrence; Research; Research Proposals; Role; Saint Jude Children' s Research Hospital; Scientist; Testing; Therapeutic; Therapeutic Intervention; Time; Training; Transcriptional Regulation; Validation; career; cell growth; cell transformation; chimeric gene; chromatin remodeling; design; drug efficacy; effective therapy; epigenomics; experimental study; exportin 1 protein; fitness; functional genomics; high risk; histone acetyltransferase; homeodomain; in vivo; leukemia; leukemogenesis; member; mouse model; novel; novel therapeutic intervention; nucleocytoplasmic transport; pharmacologic; post-doctoral training; pre-doctoral; promoter; quantitative imaging; self-renewal; small molecule; small molecule inhibitor; stem-like cell; structural biology; targeted treatment; therapeutic target; therapeutically effective; therapy resistant

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 outcome, with approximately 35% 5 year overall survival. NUP98 rearrangements lead to expression of oncogenic chimeric gene fusions involving the intrinsically disordered, 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 studies, including my own, have shown that the ability of NUP98 FOs to localize within the nucleus in membrane-less organelles, or “puncta” formed through liquid-liquid phase separation (LLPS), is necessary for transformation and deregulated gene expression phenotypes. Nevertheless, which proteins interact with NUP98 FOs in puncta and the importance of puncta formation for effective therapeutic targeting of NUP98-rearranged cells is not known. This research proposal seeks to identify the proteins found in NUP98 FO-associated puncta, uncover how puncta alter gene regulation, and determine if puncta disruption correlates with effective treatment of NUP98-rearranged cells. Aim 1 will examine the role of histone acetyltransferase (HAT) complex members, which my preliminary data identified as key NUP98 FO interacting proteins, in NUP98::KDM5A FO-driven cell transformation. I will perform CRISPR/Cas9 editing of HAT complex genes in hematopoietic stem and progenitor cells (HSPCs) from our Nup98::Kdm5a mouse model and study the in vitro and in vivo consequences of these alterations. I will also examine gene expression and chromatin remodeling in Nup98::Kdm5a HSPCs with and without HAT complex disruption. Aim 2 will determine whether effective therapeutic targeting of NUP98 FOs leads to puncta disruption. I will perform co-localization experiments for FO with proteins involved in nuclear transport and gene regulation. I will then pharmacologically inhibit these interacting proteins using available small molecule inhibitors and assess changes in puncta features and cell viability over time to determine if puncta disruption correlates with drug efficacy. In Aim 3, I will identify interacting proteins that are vulnerabilities in NUP98-rearranged cells and use pharmacologic inhibition of crucial interactors to identify how they are involved in cell transformation, gene regulation, and LLPS. Together, these studies will identify critical interacting proteins in leukemias bearing NUP98 gene fusions, examine how they contribute to leukemogenesis, and uncover how they might be targeted for therapeutic benefit.

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固有无序N-端区和一个NUP98 C-端区的基因融合 已确定的30多个伴侣基因中。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……里面 与基因调控所需的其他机制复杂，NUP98融合癌蛋白(FOS)与 许多发育基因的启动子。这导致染色质结构改变，表达增加 靶基因和异常的造血自我更新。最近的研究，包括我自己的研究表明， NUP98FOS在无膜细胞器中定位于细胞核内的能力 液-液相分离(LLP)是转化和解除基因表达调控所必需的 表型。然而，哪些蛋白质与NUP98中的FOS相互作用以及斑点的重要性 对于NUP98重排细胞的有效治疗靶向的形成尚不清楚。这项研究建议 寻求鉴定在NUP98 FO相关斑点中发现的蛋白质，揭示斑点如何改变基因调控， 并确定点状中断是否与NUP98重排细胞的有效治疗相关。目标1将 检验组蛋白乙酰转移酶(HAT)复合体成员的作用，我的初步数据确定为 在NUP98：：KDM5A FO驱动的细胞转化中，与NUP98 FO相互作用的关键蛋白。我会表演的 CRISPR/Cas9编辑我们的造血干/祖细胞中的HAT复合体基因 Nup98：：KDM5A小鼠模型，并研究这些改变在体外和体内的后果。我也会 检测有无HAT复合体的Nup98：：KDM5A HSPC的基因表达和染色质重塑 颠覆。目的2将确定NUP98 FOS的有效治疗靶向是否会导致点状中断。 我将进行FO与参与核运输和基因调控的蛋白质的共定位实验。 然后，我将使用可用的小分子抑制剂从药物上抑制这些相互作用的蛋白质 评估点状特征和细胞活力随时间的变化，以确定点状中断是否与 药效。在目标3中，我将识别相互作用的蛋白质，这些蛋白质是NUP98重排细胞和 使用对关键相互作用因子的药物抑制来确定它们如何参与细胞转化，基因 监管和有限责任公司。总之，这些研究将确定白血病中的关键相互作用蛋白 NUP98基因融合，研究它们如何促进白血病发生，并揭示它们可能成为靶点的方式 为了治疗上的好处。