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

基本信息

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
项目类别：
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

项目摘要

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 Our unpublished preliminary studies of other recurrent leukemic fusions (namely, NUP98-PHD fusions and MSI2-HOXA9, a leukemia-related chimera formed by fusing a less-studied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。大多数 NUP98-r 患者（超过 30 名）表现出不良预后和治疗失败。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 NUP98-r 融合的一种致癌机制。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)。我们对 NUP98-HOXA9（一种 AML）的研究仍不清楚。 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 结合结构域）都表明 IDR 和 LLPS 的参与因此，我们努力发现，由于异常的基因融合，许多与白血病相关的肿瘤发生。 TF 和染色质因子获得相分离诱导的 IDR 以建立 LLPS，从而赋予嵌合体因此，致癌基因表达程序的基因组靶向能力大大增强；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，这驱动侵袭性白血病形成的 IDR 和阶段机制的剖析。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）的作用。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 CTCF 独立的，代表了以前的 3D 染色质组织的未经研究的机制我们将进一步定义 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由于具有相分离能力的分子经常涉及多种反应。人类癌症和疾病，该项目的重要性和总体影响都可能很高。