The role of DNA-PKcs in DNA repair, lymphocyte development, RNA metabolism and tumor suppression

DNA-PKcs 在 DNA 修复、淋巴细胞发育、RNA 代谢和肿瘤抑制中的作用

基本信息

批准号：
10651884
负责人：
Shan Zha
金额：
$ 56.57万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
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/10651884
关键词：
Acute Affect Alanine Anemia Binding Biochemical Biogenesis Biological Assay Blood Cells Bone marrow failure C-terminal Catalytic Domain Cell Cycle Cells Cellular biology Chromosomal translocation Clustered Regularly Interspaced Short Palindromic Repeats DNA DNA Binding DNA Double Strand Break DNA Repair DNA-PKcs DNA-dependent protein kinase Data Defect Development Double Strand Break Repair Double-Stranded RNA Embryo Erythropoiesis G22P1 gene Gene Rearrangement Genetic Genetic study Genomic Instability Genomic Segment Goals Hematopoiesis Holoenzymes Human Human Cell Line Impairment In Vitro Licensing Ligation Lymphocyte Lymphoma Malignant - descriptor Malignant Neoplasms Mammals Mediating Molecular Conformation Mus Myeloid Leukemia N-terminal Nonhomologous DNA End Joining Oncogenic Pathway interactions Phase Phosphorylation Phosphotransferases Physiological Proliferating Proteins RNA RNA Binding RNA Processing RNA metabolism Regulation Ribosomal RNA Ribosomes Role Small Nucleolar RNA Stress Structure System Tail Testing Transfer RNA Translations Tumor Suppression V(D)J Recombination XRCC5 gene artemis ataxia telangiectasia mutated protein cancer cell cancer clinical trial cancer therapy ds-DNA endonuclease genotoxicity in vivo inhibitor leukemia/lymphoma live cell imaging mouse model preservation prevent protein kinase inhibitor recruit single molecule single-molecule FRET targeted cancer therapy telomere tool

项目摘要

PROJECT SUMMARY/ABSTRACT Our application focus on DNA-dependent protein kinase (DNA-PK), a DNA repair factor with newly identified role in RNA metabolism and a target of cancer therapy, and will use genetic, cell biology and single molecule approaches to dissect the role of DNA-PK during lymphoma and leukemia-genesis and therapy. Genomic instability is the hallmark of human cancer. The Non-Homologous End-Joining (NHEJ) is a major DNA double-strand breaks (DSBs) repair pathway and is required for physiological gene-rearrangements and oncogenic chromosomal translocations in developing lymphocytes. DNA-PK, composed of KU70-KU80 heterodimer (KU) and the large catalytic subunit (DNA-PKcs), is a NHEJ factor critical for both end-processing (e.g., hairpin opening) and end-ligation during NHEJ. DNA-PKcs inhibitors is in phase I/IIa clinical trials for cancer therapy. During NHEJ, KU binds to DNA ends, recruits and activates DNA-PKcs. Loss of DNA-PKcs abrogate Artemis endonuclease mediated end-processing without abolishing end-ligation. We showed that expression of kinase-dead (KD) (DNA-PKcsKD/KD) abrogates end-ligation without affecting end-processing, uncovering an end- protection role of DNA-PKcs that is regulated by its own kinase activity. End-processing in DNA-PKcsKD/KD mice is blocked by ATM inhibition, indicating end-processing requires DNA-PKcs protein, and the kinase activity from either DNA-PKcs or the related ATM kinase in vivo. DNA-PKcs is the best characterized substrate of DNA-PK and can also be phosphorylated by ATM. Mice carrying phosphorylation-deficient (DNA-PKcs5A/5A) DNA-PKcs display mild end-ligation defects and are sensitive to ATM inhibition. Thus, we propose that once assembled on KU-bound DNA, DNA-PK phosphorylation regulates end-processing and eventually the release of DNA-PKcs to licence end-ligation. Moreover, we found that Ku can also direct the assembly of DNA-PKcs on structured RNA (e.g., rRNA and snoRNA), where phosphorylation defective (DNA-PK5A) or KD DNA-PKcs (DNA-PKcsKD/KD) blocks rRNA processing, protein translation, and erythropoiesis, leading to Trp53-dependent bone marrow failure. These findings uncovered a NHEJ-independent role of DNA-PK in mammals. And two-third of DNA- PKcsKD/KDTrp53-/- mice succumbed to ribosomal stress induced myeloid leukemia and one-third died of lymphomas with IgH-Myc translocations, highlighting the critical role of DNA-PK in tumor suppression. Based on these and other findings, we hypothesize that DNA-PKcs kinase activity and auto-phosphorylation regulates KU-dependent assembly of DNA-PK on DNA and RNA to suppress lymphoma and leukemia genesis. To test this, we will 1) characterize and compare KU and DNA-PK dynamics on RNA vs DNA; 2) elucidate how KU- depletion impact RNA processing in human cells; 3) determine the physiological function of KU80 C-terminal domain and tail in lymphoma and leukemia genesis and the recruitment and stabilization of DNA-PKcs. The results will reveal the regulation and function of the RNA & DNA dependent function of DNA-PK, the essential role of KU in human cells, and the broad impacts of DNA-PK inhibition.

项目概要/摘要我们的应用重点是 DNA 依赖性蛋白激酶 (DNA-PK)，这是一种新的 DNA 修复因子确定了 RNA 代谢中的作用和癌症治疗的目标，并将利用遗传、细胞生物学和单一方法分子方法剖析 DNA-PK 在淋巴瘤和白血病发生和治疗过程中的作用。基因组不稳定是人类癌症的标志。非同源末端连接 (NHEJ) 是 DNA 的主要组成部分双链断裂（DSB）修复途径，是生理基因重排和发育中的淋巴细胞中的致癌染色体易位。 DNA-PK，由 KU70-KU80 组成异二聚体 (KU) 和大催化亚基 (DNA-PKcs) 是对末端加工至关重要的 NHEJ 因子（例如，发夹打开）和 NHEJ 期间的末端结扎。 DNA-PKcs 抑制剂正在进行 I/IIa 期癌症临床试验治疗。在 NHEJ 期间，KU 与 DNA 末端结合，招募并激活 DNA-PKc。 DNA-PKcs 丢失会废除 Artemis 核酸内切酶介导末端加工而不废除末端连接。我们展示了这个表达式激酶死亡 (KD) (DNA-PKcsKD/KD) 废除末端连接而不影响末端处理，从而揭示末端连接 DNA-PKcs 的保护作用受其自身激酶活性的调节。 DNA-PKcsKD/KD 小鼠的末端加工被 ATM 抑制所阻断，表明末端加工需要 DNA-PKcs 蛋白，并且激酶活性来自 DNA-PKcs 或体内相关的 ATM 激酶。 DNA-PKcs 是 DNA-PK 最具特征的底物也可以被ATM磷酸化。携带磷酸化缺陷 (DNA-PKcs5A/5A) DNA-PKcs 的小鼠显示出轻微的末端连接缺陷并且对 ATM 抑制敏感。因此，我们建议一旦组装 KU 结合的 DNA、DNA-PK 磷酸化调节末端加工并最终释放 DNA-PKcs 许可末端连接。此外，我们发现Ku还可以指导DNA-PKcs在结构化RNA上的组装（例如，rRNA 和 snoRNA），其中磷酸化缺陷 (DNA-PK5A) 或 KD DNA-PKcs (DNA-PKcsKD/KD) 阻断 rRNA 加工、蛋白质翻译和红细胞生成，导致 Trp53 依赖性骨髓衰竭。这些发现揭示了 DNA-PK 在哺乳动物中的独立于 NHEJ 的作用。还有三分之二的DNA—— PKcsKD/KDTrp53-/- 小鼠死于核糖体应激诱导的骨髓性白血病，其中三分之一死于具有 IgH-Myc 易位的淋巴瘤，强调了 DNA-PK 在肿瘤抑制中的关键作用。基于这些和其他发现，我们假设 DNA-PKcs 激酶活性和自磷酸化调节 DNA-PK 在 DNA 和 RNA 上的 KU 依赖性组装可抑制淋巴瘤和白血病的发生。到对此进行测试，我们将 1) 表征并比较 RNA 与 DNA 上的 KU 和 DNA-PK 动态； 2）阐明如何KU- 耗竭影响人体细胞中的 RNA 加工； 3)确定KU80 C端的生理功能淋巴瘤和白血病发生以及 DNA-PKc 的募集和稳定中的结构域和尾部。这结果将揭示 DNA-PK 的 RNA 和 DNA 依赖性功能的调节和功能，DNA-PK 是必需的 KU 在人类细胞中的作用，以及 DNA-PK 抑制的广泛影响。