Phase separation-induced nuclear organization in ALT Cancer

相分离诱导的 ALT 癌症核组织

基本信息

批准号：
10117580
负责人：
Huaiying Zhang
金额：
$ 53.09万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-10 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10117580
关键词：
3-Dimensional Acute Promyelocytic Leukemia Address Adopted Behavior Biological Markers Body Composition Cancer Cell Growth Cell Nucleus Cell Proliferation Cell physiology Cells Chromosome Structures Cultured Cells DNA Repair DNA biosynthesis Diagnosis Exhibits Gene Expression Genome Human Immune response Knowledge Label Length Liquid substance Malignant Neoplasms Mechanics Microscopy Modeling Molecular Monitor Nature Nuclear Phase Phenotype Physical condensation Process Proteins RNA RNA-Binding Proteins RNA-Directed DNA Polymerase Resolution Structure Telomerase Telomerase inhibition Telomere Maintenance Telomere Pathway Testing Tissues Untranslated RNA base cancer cell cancer therapy homologous recombination insight novel therapeutics optogenetics prevent recruit repaired side effect single molecule telomere therapeutic development therapeutic target tool

项目摘要

Abstract: All cancer cells need to maintain telomere length for immortality. While most cancer cells reactivate telomerase, a reverse transcriptase, to elongates telomere from an RNA template, about 10-15% of cancer cells are telomerase-negative and adopt a homologous-recombination based alternative lengthening of telomeres (ALT) pathway. ALT cells exhibit many abnormalities in nuclear organization, including the formation of nuclear bodies called APBs for ALT telomere-associated promyelocytic leukemia nuclear bodies, clustering of telomeres within APBs, and the formation of RNA foci on telomeres with a long non-coding RNA called telomere repeat-containing RNA (TERRA). These unique features are used as biomarkers for ALT diagnosis and can be attractive therapeutic targets because of reduced side effects on healthy cells that do not share these features. However, how these features contribute to telomere maintenance and ALT cancer cell growth remain elusive, due to the lack of conceptual model as well as experimental tools to monitor and control their assembly and function in live cells. Based on our observation that APBs exhibit liquid behavior and long non- coding RNAs can phase separate with RNA-binding proteins, we propose a liquid-liquid phase separation model for the assmembly and function of these ALT specific features. We hypothesize TERRA phase separates with its interacting proteins to nucleate APB liquid droplets. The liquid nature of APBs droplets (also called condensates) would promote coalescence of APBs to drive telomere clustering. Meanwhile, condensation of APB droplets can concentrate DNA repair factors, providing opportunities for telomeres to use one another as repair templates to elongate within APBs. To test our hypothesis, we developed a state-of-the- art optogenetic approach to control APB assembly. We demonstrate that liquid phase separation underlies APB assembly and coalescence of APB droplets indeed drives telomere clustering. Building on our ability to control telomere clustering and APB assembly and by collaborating with experts in super resolution microscopy, nuclear mechanics, chromosome organization and ALT cancer, we will investigate how DNA repair factors are recruited to and organized in APB condensates for ALT telomere DNA synthesis (Aim 1) and how telomere clustering leads to unique genome organization and gene expression in ALT cells (Aim 2). We will then extend our optogenetic tools to control RNA and dissect TERRA contributions in ALT (Aim 3). Results obtained by manipulating cultured ALT cells will be confirmed by characterizing ALT tissue or creating de novo ALT phonotypes in primary human cells. Our results will provide mechanistic understanding on how protein and/or RNA phase separation contributes to ALT cancer, which will offer the potential to develop strategies specifically targeting these unique phase separation processes, rather than the existing molecules that shared by heathy cells, for ALT cancer treatment. Such therapies are also beneficial for treating telomerase-positive cancer as these cancer cells can escape telomerase inhibition and adopt ALT for telomere maintenance.

摘要：所有癌细胞都需要维持端粒长度才能永生。虽然大多数癌细胞会重新激活端粒酶是一种逆转录酶，可从 RNA 模板延长端粒，约占癌症的 10-15% 细胞是端粒酶阴性的，并采用基于同源重组的替代延长端粒 (ALT) 途径。 ALT 细胞在核组织中表现出许多异常，包括形成称为 APB 的核体，用于 ALT 端粒相关早幼粒细胞白血病核体、聚类 APB 内端粒的形成，以及端粒上 RNA 焦点的形成，其中带有长非编码 RNA，称为含有端粒重复序列的 RNA (TERRA)。这些独特的特征被用作 ALT 诊断的生物标志物由于对健康细胞的副作用减少，因此可以成为有吸引力的治疗靶点这些功能。然而，这些特征如何有助于端粒维持和 ALT 癌细胞生长由于缺乏概念模型以及监测和控制其的实验工具，仍然难以捉摸。活细胞中的组装和功能。根据我们的观察，APB 表现出液体行为和长期非编码RNA可以与RNA结合蛋白进行相分离，我们提出了液-液相分离这些 ALT 特定功能的组装和功能模型。我们假设 TERRA 阶段与其相互作用的蛋白质分离，使 APB 液滴成核。 APB 液滴的液体性质（也称为冷凝物）会促进 APB 的聚结，从而驱动端粒聚集。同时， APB液滴的凝结可以浓缩DNA修复因子，为端粒提供利用机会彼此作为修复模板在 APB 内延伸。为了检验我们的假设，我们开发了一个最新的- 艺术光遗传学方法来控制 APB 组装。我们证明液相分离是 APB 组装和 APB 液滴的聚结确实驱动端粒聚集。以我们的能力为基础控制端粒聚类和 APB 组装，并与超分辨率专家合作显微镜、核力学、染色体组织和 ALT 癌症，我们将研究 DNA 如何修复因子被招募并组织在 APB 凝聚体中，用于 ALT 端粒 DNA 合成（目标 1）和端粒聚类如何导致 ALT 细胞中独特的基因组组织和基因表达（目标 2）。我们然后将扩展我们的光遗传学工具来控制 RNA 并剖析 TERRA 在 ALT 中的贡献（目标 3）。结果通过操作培养的 ALT 细胞获得的结果将通过表征 ALT 组织或从头创建来确认原代人类细胞中的 ALT 表型。我们的结果将为蛋白质如何和/或 RNA 相分离导致 ALT 癌症，这将提供开发策略的潜力专门针对这些独特的相分离过程，而不是共享的现有分子由健康细胞产生，用于 ALT 癌症治疗。此类疗法也有利于治疗端粒酶阳性的患者癌症，因为这些癌细胞可以逃避端粒酶抑制并采用 ALT 来维持端粒。