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

基本信息

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
项目类别：
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&apos 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

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 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），与对治疗和预后不良的抗药性有关大约35％5年的总生存期。 NUP98重排导致致癌嵌合体的表达涉及NUP98的内在无序的N末端区域的基因融合和一个融合超过30个确定的伴侣基因。伴侣基因通常具有具有关键功能特性的域，包括同源域部分（例如HOXA9）和在转录调节中的角色（例如NSD1，KDM5A）。与基因调节所需的其他机械复合物，NUP98融合癌蛋白（FOS）与许多发育基因的启动子。这导致染色质结构的变化，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 NUP98 FOS在无膜细胞器中核中定位的能力，或通过形成的“点” 液态液相分离（LLP）是转化和放松调节基因表达所必需的表型。然而，哪种蛋白质与点的NUP98 FOS相互作用以及Puncta的重要性尚不清楚用于有效的NUP98重新培养细胞的治疗靶向的形成。这项研究建议旨在鉴定NUP98 FO相关的点中发现的蛋白质，揭示了点状调节基因调节，并确定PuncTA破坏是否与NUP98重建细胞的有效治疗相关。目标1意志检查组蛋白乙酰转移酶（HAT）复合构件的作用，我的初步数据将其确定为 NUP98 :: KDM5A FO驱动的细胞转换中的关键NUP98 FO相互作用蛋白。我会表演造血茎和祖细胞（HSPC）中HAT复合基因的CRISPR/CAS9编辑我们 NUP98 :: KDM5A小鼠模型并研究这些改变的体外和体内后果。我也会在NUP98中检查基因表达和染色质重塑，hspcs带有和没有帽子复合物破坏。 AIM 2将确定NUP98 FOS的有效治疗靶向是否会导致点击中断。我将与涉及核转运和基因调节的蛋白质进行共定位实验。然后，我将使用可用的小分子抑制剂和评估点状特征和细胞活力随时间的变化，以确定点的破坏是否与药物效率。在AIM 3中，我将确定在NUP98重新传输细胞中脆弱性的相互作用蛋白质和使用对关键相互作用者的药物通言抑制，以确定它们如何参与细胞转化，基因调节和LLP。这些研究将共同确定白血病中的关键相互作用蛋白 NUP98基因融合，检查它们如何对白血病发生贡献，并发现它们如何被靶向为了治疗益处。