Phase separation-induced nuclear organization in ALT Cancer

相分离诱导的 ALT 癌症核组织

• 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
• 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

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：所有癌细胞都需要保持端粒长度以使其永生。而大多数癌细胞重新激活 端粒酶（一种逆转录酶）从RNA模板中拉长端粒，约有10-15％ 细胞是端粒酶阴性的 端粒（ALT）途径。 ALT细胞在核组织中表现出许多异常，包括形成 核体称为APB的ALT端粒相关的Promyelocytic Leukemia核体，聚类 APB中的端粒的形成，RNA焦点在端粒上具有长长的非编码RNA 端粒重复的RNA（Terra）。这些独特的功能被用作ALT诊断的生物标志物 并且由于对健康细胞的副作用减少而可能是有吸引力的治疗靶标 这些功能。但是，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视和控制其的实验工具，保持难以捉摸 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结合蛋白分离，我们提出了液态液相的分离 这些ALT特定特征的辅助和功能的模型。我们假设Terra阶段 与其相互作用的蛋白分离为成核APB液滴。 APB液滴的液体性质（也是 称为冷凝物）将促进APB的合并以驱动端粒聚类。同时， APB液滴的凝结可以集中DNA修复因子，为端粒提供了机会 彼此作为修复模板在APB中伸长的模板。为了检验我们的假设，我们开发了 控制APB组装的ART遗传方法。我们证明了液相分离的基础 APB液滴的APB组装和聚合确实驱动了端粒聚类。以我们的能力为基础 控制端粒聚类和APB组件，并通过与超级分辨率的专家合作 显微镜，核力学，染色体组织和ALT癌，我们将研究DNA 修复因子被募集到APB冷凝水中，以进行Alt端粒DNA合成（AIM 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（AIM 2）。我们 然后，将扩展我们的光遗传学工具以控制RNA并在ALT中剖析Terra贡献（AIM 3）。结果 通过操纵培养的Alt细胞获得的将通过表征Alt组织或创建从头开始的确认 原代人细胞中的Alt Phonotypes。我们的结果将提供有关蛋白质如何的机械理解 和/或RNA期分离有助于ALT癌，这将提供制定策略的潜力 专门针对这些独特的相分离过程，而不是共享的现有分子 通过Heaty细胞进行ALT癌症治疗。这种疗法也有益于治疗端粒酶阳性 癌症作为这些癌细胞可以避免端粒酶抑制，并采用ALT进行端粒维持。