Identification of Essential Factors for the Alternative Lengthening of Telomeres Pathway

端粒途径选择性延长的关键因素的鉴定

• 批准号: 9891984
• 负责人: Taylor Kristine Loe
• 金额: $ 0.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9891984
• 关键词: Acute Promyelocytic Leukemia; Address; Anemia; Apoptosis; Binding Proteins; Bypass; CRISPR/Cas technology; Cell Aging; Cell Line; Cells; Chromosomes; Complex; DNA; DNA Damage; DNA Repair; Ensure; Enzymes; FANCD2 protein; Fanconi Anemia Complementation Group A Protein; Genetic; Genetic Recombination; Genome Stability; Guide RNA; Human; Investigation; Knock-out; Lead; Length; Light; Maintenance; Malignant Neoplasms; Names; Nuclear; Pathway interactions; Proteins; RNA; RNA-Directed DNA Polymerase; Role; Suggestion; Telomerase; Telomere Maintenance; Telomere Pathway; Telomere Recombination; Telomere Shortening; Topoisomerase; Topoisomerase III; alternative treatment; cancer cell; endonuclease; genome editing; genome wide screen; genome-wide; homologous recombination; novel; protein complex; telomere; therapeutic target; transcription factor PML

ABSTRACT Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural ends of linear chromosomes and ensure genome stability. Due to the end replication problem, telomeres progressively shorten with each cellular division until they become critically short, resulting in cellular senescence or apoptosis. A universal feature of human cancer cells is the ability to bypass this proliferation barrier by engaging a telomere elongation mechanism, which is most often the expression of the reverse transcriptase telomerase. However, a significant portion of cancer cells (approx 10-15%) utilize a telomerase-independent pathway named the Alternative Lengthening of Telomeres (ALT) pathway that relies on homologous recombination between telomeric sequences to extend telomeres and evade replicative arrest. Cancer cells that utilize ALT are characterized by heterogeneous telomere lengths, extrachromosomal telomeric DNA and the presence of promyelocytic leukemia (PML) bodies at telomeres. Although the ALT pathway has been the subject of intense investigation, the mechanism by which it occurs is still incompletely understood. In order to shed light on the ALT pathway, we will employ CRISPR/Cas9 to generate genetic knockouts of the known ALT-associated factors PML and SUMO2 to assess their essentiality in ALT activity. Additionally, we will use CRISPR/Cas9 to perform a genome-wide gRNA knockout screen to identify novel factors required for ALT. In this way, we will identify factors essential for ALT to expand our understanding of this telomere maintenance mechanism and reveal potential therapeutic targets for the treatment of ALT cancers.

摘要 端粒由蛋白质复合物结合的TTAGGG重复序列组成，蛋白质复合物用于保护自然细胞， 线性染色体的末端，并确保基因组的稳定性。由于端复制问题， 端粒随着每次细胞分裂而逐渐缩短，直到它们变得非常短， 在细胞衰老或凋亡中的作用。人类癌细胞的普遍特征是能够绕过 这种增殖障碍通过参与端粒延长机制，这是最常见的 逆转录酶端粒酶的表达。然而，很大一部分癌细胞 （约10-15%）利用一种称为替代性端粒延长的端粒非依赖性途径。 端粒（ALT）途径依赖于端粒序列之间的同源重组， 延长端粒并逃避复制停滞。利用ALT的癌细胞的特征在于： 端粒长度不均匀，染色体外端粒DNA和存在 早幼粒细胞白血病（PML）的身体在端粒。虽然ALT途径一直是 尽管进行了大量的研究，但其发生的机制仍不完全清楚。为了 为了阐明ALT途径，我们将使用CRISPR/Cas9来产生ALT途径的基因敲除。 已知ALT相关因子PML和SUMO 2，以评估其在ALT活性中的重要性。 此外，我们将使用CRISPR/Cas9进行全基因组gRNA敲除筛选， ALT所需的新因素。通过这种方式，我们将确定ALT扩展我们的 了解这种端粒维持机制，并揭示潜在的治疗靶点， ALT癌症的治疗。