Phase separation-induced nuclear organization in ALT Cancer

摘要：所有的癌细胞都需要保持端粒的长度才能永生。当大多数癌细胞重新激活时 端粒酶，一种逆转录酶，从RNA模板拉长端粒，约占癌症的10%-15% 细胞是端粒酶阴性的，采用基于同源重组的交替延长 端粒(ALT)途径。ALT细胞在核组织中表现出许多异常，包括形成 ALT端粒相关早幼粒白血病核体的APB，聚集性 在APB内的端粒，以及在端粒上形成RNA焦点的长非编码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 可以成为有吸引力的治疗靶点，因为减少了对健康细胞的副作用 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 仍然难以捉摸，因为缺乏概念模型以及监测和控制其 在活细胞中组装和发挥功能。根据我们的观察，APB表现出液体行为和长期的非 编码的RNA可以与RNA结合蛋白相分离，我们提出了液-液相分离 型号为ALT的分类和功能的具体功能。我们假设了Terra阶段 与其相互作用的蛋白质分离，形成APB液滴。APBS液滴的液体性质(也 称为缩合物)将促进APB的结合以驱动端粒聚集。同时， APB液滴的凝聚可以浓缩DNA修复因子，为端粒提供使用机会 彼此作为修复模板以在APB内伸长。为了检验我们的假设，我们开发了一个状态- 控制APB组装的ART光遗传学方法。我们证明了液体相分离是 APB液滴的APB组装和聚合确实推动了端粒聚集。以我们的能力为基础 控制端粒聚集和APB组装，并通过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 修复因子被招募到APB冷凝物中并在其中组织，以合成ALT端粒DNA(目标1)和 端粒聚集如何导致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工具来控制RNA并剖析Terra对ALT的贡献(目标3)。结果 通过操纵培养的ALT细胞获得的将通过表征ALT组织或创造新的 人类原代细胞中的ALT音型。我们的结果将提供对蛋白质如何 和/或RNA时相分离有助于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 通过健康细胞，用于治疗ALT癌症。这样的疗法对治疗端粒酶阳性也是有益的。 癌症，因为这些癌细胞可以逃脱端粒酶的抑制，并采用ALT来维持端粒。

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.