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)，与治疗耐药和不良预后相关，大约 35% 的 5 年总生存率 NUP98 重排导致致癌嵌合体的表达。涉及本质上无序的 NUP98 N 端区域和 NUP98 的 C 端区域的基因融合超过 30 个已识别的伙伴基因伙伴基因通常具有具有关键功能特性的域，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）。 NUP98 融合癌蛋白 (FO) 与基因调控所需的其他机制形成复合体，与许多发育基因的启动子这会导致染色质结构的变化，增加表达。最近的研究，包括我自己的研究表明， NUP98 FO 定位在无膜细胞器核内的能力，或通过形成的“点” 液-液相分离（LLPS）对于转化和解除基因表达调控是必要的尽管如此，哪些蛋白质与点中的 NUP98 FO 相互作用以及点的重要性。 NUP98 重排细胞的有效治疗靶向的形成尚不清楚。试图鉴定 NUP98 FO 相关 puncta 中发现的蛋白质，揭示 puncta 如何改变基因调控，并确定泪点破坏是否与 NUP98 重排细胞的有效治疗相关，目标 1。检查组蛋白乙酰转移酶 (HAT) 复合体成员的作用，我的初步数据将其确定为我将在 NUP98::KDM5A FO 驱动的细胞转化中执行关键的 NUP98 FO 相互作用蛋白。对我们的造血干细胞和祖细胞 (HSPC) 中的 HAT 复合体基因进行 CRISPR/Cas9 编辑我还将研究 Nup98::Kdm5a 小鼠模型并研究这些改变的体外和体内后果。检查带有和不带有 HAT 复合物的 Nup98::Kdm5a HSPC 中的基因表达和染色质重塑目标 2 将确定 NUP98 FO 的有效治疗靶向是否会导致泪点破坏。我将对 FO 与参与核运输和基因调控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从药理学上抑制这些相互作用的蛋白质评估泪点特征和细胞活力随时间的变化，以确定泪点破坏是否与在目标 3 中，我将识别 NUP98 重排细胞中的脆弱性相互作用蛋白和使用关键相互作用物的药理抑制来确定它们如何参与细胞转化、基因这些研究将共同鉴定白血病携带中的关键蛋白质。 NUP98 基因融合，检查它们如何促进白血病发生，并揭示它们如何被靶向以获得治疗效果。