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: 10539807
• 负责人: Douglas H. Phanstiel
• 金额: $ 57.78万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10539807
• 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; 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; PHD Finger; Patients; Phase; Phenotype; Phenylalanine; Plants; Prognosis; Protein Region; Proteins; Proto-Oncogenes; Public Health; RNA-Binding Proteins; Recurrence; Research; Role; Treatment Failure; base; 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.

项目摘要/摘要 NUP98基因（NUP98-R）的重排在白血病中是复发的，例如急性髓样白血病（AML）。 NUP98-R患者的预后和治疗衰竭状况不佳。大多数NUP98-R合作伙伴（从 患者）是转录因子的DNA结合域（TF； Hoxa9）或Hisstone结合基序，例如 植物同源域（PHD），表明染色质失调是一种致癌机制。 NUP98-R融合 NUP98的苯丙氨酸 - 甘氨酸（FG）重复始终保留，称为本质上受干扰区域（IDR）。 非结构化的IDR如何导致肿瘤发生仍然难以捉摸。我们对AML NUP98-HOXA9的研究 NUP98-TF嵌合体，揭示NUP98 IDR对液体液相分离（LLP）的基本要求。 我们还表明，IDR和LLP对于NUP98-HOXA9和 对于癌基因启动子和增强剂之间的长距离染色质循环，导致 小鼠的侵略性AML。我们对其他复发性白血病融合的未发表的初步研究（即 NUP98-PHD融合和MSI2-HOXA9，这是一种与白血病相关的嵌合体，该嵌合体通过融合不太研究的IDR形成 带有HOXA9的DNA结合域的RNA结合蛋白）所有指向IDR和LLP的参与 肿瘤发生。这就是我们假设，由于异常的基因融合，许多与白血病有关的Onco- TF和染色质因子获得了诱导相位分离的IDR来建立LLP，该LLP供认嵌合体 基因组靶向的能力更加增强；因此，致癌基因表达程序是 在癌基因启动子和增强子之间形成异常染色质环时，过度激活的同时， 驱动侵略性白血病的形成。解剖IDR和相位的机制 分离介导的异常基因组组织和癌细胞中的癌基因激活应提供新的 以及范式转移观点，即对侵略性癌症的发展（暗示可能是新疗法） 将来。为了实现这一目标，我们将进一步描述未经研究的IDR的角色（MSI2的角色） 在体外和细胞中建立LLP（AIM 1A），并将使用原代人造血茎/祖细胞 细胞（HSPC）和衍生细胞定义IDR和LLP在调节基因组靶向（1B）中的作用（1B） 靶基因表达（1C）和白血病在体外/体内（1D）通过各种融合（NUP98-PHD） 和MSI2/NUP98-HOXA9）。 LLPS指定的染色质环路是CTCF无关的，代表了先前的 3D染色质组织的未研究机制。我们将进一步定义3D染色质结构 由疾病相关细胞中各种NUP98-R和MSI2-HOXA9融合引起的改变（AIM 2A），定义了 分子机制驱动LLPS依赖性环（2B）的形成/维护，并确定 LLP DNA回路对使用新型CRISPR/DCAS9-IDR的影响对癌基因的持续激活的影响 融合策略（2C）。由于相分开能力的分子经常在广泛的范围内实施 该项目的意义和整体影响都可能很高。