The role for phase separation in oncogenesis and aberrant chromatin looping formation

相分离在肿瘤发生和异常染色质环形成中的作用

• 批准号: 10908136
• 负责人: Douglas H. Phanstiel
• 金额: $ 57.28万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-04 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10908136
• 关键词: 3-Dimensional; Acute Myelocytic Leukemia; Automobile Driving; Behavior Control; Binding; Biological Models; Cells; Chimera organism; Chromatin; Chromatin Loop; Chromatin Structure Alteration; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Binding; DNA Binding Domain; Development; Disease; Dissection; Enhancers; Exhibits; Fingers; Fusion Oncogene Proteins; Future; Gene Expression; Gene Fusion; Genes; Genetic Transcription; Genome; Genomics; Glycine; Goals; HOXA9 gene; Hematology; Hematopoietic Neoplasms; Hematopoietic stem cells; Histones; Human; In Vitro; Leukemic Cell; Liquid substance; Maintenance; Malignant Neoplasms; Mediating; Modeling; Molecular; Mus; N-terminal; Nuclear Pore Complex Proteins; Oncogene Activation; Oncogenes; Oncogenic; Oncoproteins; Outcome; Patients; Phase; Phenotype; Phenylalanine; Physical condensation; Plants; Prognosis; Protein Region; Proteins; Proto-Oncogenes; Public Health; RNA-Binding Proteins; Recurrence; Research; Role; Treatment Failure; acquired factor; cancer cell; homeodomain; insight; leukemia; leukemic transformation; leukemogenesis; novel; novel therapeutic intervention; programs; promoter; transcription factor; tumor; tumorigenesis

PROJECT SUMMARY/ABSTRACT Rearrangement of NUP98 gene (NUP98-r) is recurrent in leukemias such as acute myeloid leukemia (AML). Patients with NUP98-r show poor prognosis and therapy failure. Most NUP98-r partners (>30 identified from patients) are a DNA-binding domain of transcription factor (TF; e.g. HOXA9) or a histone-binding motif such as Plant Homeodomain (PHD), suggesting chromatin deregulation as an oncogenic mechanism. NUP98-r fusions invariably retain Phenylalanine-Glycine (FG) repeats, termed intrinsically disordered region (IDR), from NUP98. How unstructured IDR contributes to oncogenesis remains elusive. Our studies of NUP98-HOXA9, an AML NUP98-TF chimera, unveil an essential requirement of NUP98’s IDR for liquid-liquid phase separation (LLPS). We also show that IDR and LLPS are critical for the much-enhanced genome binding by NUP98-HOXA9 and for long-distance chromatin looping between oncogene promoters and enhancers, leading to development of aggressive AML in mice. Our unpublished preliminary studies of other recurrent leukemic fusions (namely, NUP98-PHD fusions and MSI2-HOXA9, a leukemia-related chimera formed by fusing a less-studied IDR of an RNA-binding protein with HOXA9’s DNA-binding domain) all point to involvements of IDR and LLPS for oncogenesis. Thus, we hypothesize that, due to aberrant genic fusions, a number of leukemia-related onco- TFs and chromatin factors acquire a phase-separation-inducing IDR to establish LLPS, which confers chimera a much more enhanced ability in genomic targeting; consequently, an oncogenic gene-expression program is over-activated while aberrant chromatin loops are formed between oncogene promoters and enhancers, which drives formation of aggressive leukemias. Dissection of the mechanisms underlying the IDR- and phase- separation-mediated aberrant genome organization and oncogene activation in cancer cells shall provide new and paradigm-shifting views as for how aggressive cancer develops, implicative of potentially new treatments in future. Towards this goal, we will further characterize the role for the un-studied IDR (that of MSI2) in establishing LLPS in vitro and in cells (Aim 1A) and will use primary human hematopoietic stem/progenitor cells (HSPCs) and derived cells to define roles of IDR and LLPS in regulating genomic targeting (1B), the target gene expression (1C), and leukemic transformation in vitro/vivo (1D) by various fusions (NUP98-PHD and MSI2/NUP98-HOXA9). LLPS-indued chromatin looping is CTCF-independent and represents a previously unstudied mechanism underlying 3D chromatin organization. We will further define the 3D chromatin structure alterations caused by various NUP98-r and MSI2-HOXA9 fusions in disease-relevant cells (Aim 2A), define the molecular mechanisms driving formation/maintenance of LLPS-dependent loops (2B), and determine the impact of LLPS DNA loops on the sustained activation of oncogenes by using a novel CRISPR/dCas9-IDR fusion strategy (2C). As phase-separation-competent molecules are frequently implicated in a wide range of human cancers and diseases, both the significance and overall impact of the project are potentially high.

项目概要/摘要 NUP98 基因 (NUP98-r) 重排在急性髓系白血病 (AML) 等白血病中反复出现。 NUP98-r 患者预后不良且治疗失败。大多数 NUP98-r 合作伙伴（超过 30 个来自 患者）是转录因子（TF；例如 HOXA9）的 DNA 结合结构域或组蛋白结合基序，例如 植物同源结构域 (PHD)，表明染色质失调是一种致癌机制。 NUP98-r 融合 始终保留来自 NUP98 的苯丙氨酸-甘氨酸 (FG) 重复序列，称为本质无序区域 (IDR)。 非结构化 IDR 如何促进肿瘤发生仍然难以捉摸。我们对 NUP98-HOXA9（一种 AML）的研究 NUP98-TF 嵌合体揭示了 NUP98 IDR 对液-液相分离 (LLPS) 的基本要求。 我们还表明，IDR 和 LLPS 对于 NUP98-HOXA9 和 NUP98-HOXA9 显着增强的基因组结合至关重要。 癌基因启动子和增强子之间的长距离染色质环，导致发展 小鼠的侵袭性 AML。我们未发表的其他复发性白血病融合的初步研究（即， NUP98-PHD 融合体和 MSI2-HOXA9，一种通过融合较少研究的 IDR 形成的白血病相关嵌合体 RNA 结合蛋白与 HOXA9 的 DNA 结合域）都表明 IDR 和 LLPS 的参与 肿瘤发生。因此，我们假设，由于异常的基因融合，许多与白血病相关的肿瘤 TF 和染色质因子获得相分离诱导的 IDR 以建立 LLPS，从而赋予嵌合体 基因组靶向能力大大增强；因此，致癌基因表达程序是 过度激活，而癌基因启动子和增强子之间形成异常染色质环，这 促进侵袭性白血病的形成。剖析 IDR 和阶段的机制 癌细胞中分离介导的异常基因组组织和癌基因激活将提供新的 关于侵袭性癌症如何发展的范式转变观点，暗示潜在的新疗法 未来。为了实现这一目标，我们将进一步描述未研究的 IDR（MSI2）在 在体外和细胞内建立 LLPS（目标 1A）并将使用原代人类造血干/祖细胞 细胞 (HSPC) 和衍生细胞来定义 IDR 和 LLPS 在调节基因组靶向中的作用 (1B)、 靶基因表达 (1C)，以及通过各种融合 (NUP98-PHD) 进行的体外/体内白血病转化 (1D) 和 MSI2/NUP98-HOXA9)。 LLPS 引起的染色质环是不依赖于 CTCF 的，并且代表了先前的 3D 染色质组织的潜在机制尚未研究。我们将进一步定义3D染色质结构 疾病相关细胞中各种 NUP98-r 和 MSI2-HOXA9 融合引起的改变（目标 2A），定义 驱动 LLPS 依赖环 (2B) 形成/维持的分子机制，并确定 使用新型 CRISPR/dCas9-IDR LLPS DNA 环对癌基因持续激活的影响 融合策略（2C）。由于具有相分离能力的分子经常涉及多种 人类癌症和疾病，该项目的重要性和总体影响都可能很高。