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

• 批准号: 10539944
• 负责人: Shan Zha
• 金额: $ 59.75万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 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/10539944
• 关键词: Acute; Affect; Alanine; Anemia; Binding; Biochemical; Biogenesis; Biological Assay; Bone marrow failure; C-terminal; Catalytic Domain; Cell Cycle; Cells; Cellular biology; Chromosomal translocation; Clustered Regularly Interspaced Short Palindromic Repeats; Cyclic AMP-Dependent Protein Kinases; 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; Gene Rearrangement; Genetic; Genetic study; Genomic Instability; Genomic Segment; Goals; Holoenzymes; Human; Human Cell Line; Impairment; In Vitro; Licensing; Ligation; Lymphocyte; Lymphoma; Malignant Neoplasms; Mammals; Mediating; Molecular Conformation; Mus; Myeloid Leukemia; N-terminal; Nonhomologous DNA End Joining; Oncogenic; Pathway interactions; Phase; Phosphorylation; Phosphotransferases; Physiological; Proliferating; Protein Kinase; 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; base; cancer cell; cancer clinical trial; cancer therapy; ds-DNA; endonuclease; genotoxicity; in vivo; inhibitor; leukemia/lymphoma; live cell imaging; mouse model; preservation; protein kinase inhibitor; recruit; single molecule; single-molecule FRET; targeted cancer therapy; telomere; tool

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) 是一个主要的 DNA 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) 取消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，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。