Functional and Pharmacologic Investigation of the NUP98 Fusion Oncoprotein Interactome

项目摘要 在大约5%的儿童中观察到涉及核孔蛋白98（NUP 98）的染色体易位。 急性髓性白血病（AML），与治疗抵抗和不良结局相关， 约35%的5年总生存率。NUP 98重排导致致癌嵌合基因的表达 涉及NUP 98的固有无序的N-末端区域和NUP 98的C-末端区域的基因融合体， 超过30个已识别的伴侣基因。伴侣基因通常具有关键功能特性的结构域， 包括同源结构域部分（例如HOXA 9）和在转录调节中的作用（例如NSD 1、KDM 5A）。在 NUP 98融合癌蛋白（FO）与基因调控所需的其他机制相结合， 许多发育基因的启动子。这导致染色质结构的变化， 靶基因和异常造血自我更新。最近的研究，包括我自己的研究，表明 NUP 98 FO定位于细胞核内无膜细胞器中的能力，或通过 液-液相分离（LLPS）是转化和解除基因表达调控所必需的 表型。然而，在斑点中哪些蛋白质与NUP 98 FO相互作用以及斑点的重要性， NUP 98重排细胞的有效治疗靶向的形成是未知的。本研究提案 试图鉴定NUP 98 FO相关斑点中发现的蛋白质，揭示斑点如何改变基因调控， 并确定斑点破坏是否与NUP 98重排细胞的有效治疗相关。目标1将 研究组蛋白乙酰转移酶（HAT）复合物成员的作用，我的初步数据确定为 关键NUP 98 FO相互作用蛋白，在NUP 98：：KDM 5A FO驱动的细胞转化中。我必坚定 CRISPR/Cas9编辑来自我们的造血干细胞和祖细胞（HSPC）中的HAT复合物基因 Nup 98：：Kdm 5a小鼠模型，并研究这些改变的体外和体内后果。我也会 检查具有和不具有HAT复合物的Nup 98：：Kdm 5a HSPC中的基因表达和染色质重塑 破坏目的2将确定NUP 98 FO的有效治疗靶向是否导致斑点破坏。 我将进行FO与参与核转运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来抑制这些相互作用的蛋白质， 评估斑点特征和细胞活力随时间的变化，以确定斑点破坏是否与 药效在目标3中，我将识别NUP 98重排细胞中的相互作用蛋白质， 使用关键相互作用物的药理学抑制，以确定它们如何参与细胞转化、基因 法规和LLPS。总之，这些研究将确定关键的相互作用蛋白质在白血病轴承 NUP 98基因融合，研究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。

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.