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：
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
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/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

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.

项目摘要/摘要我们的应用专注于DNA依赖性蛋白激酶（DNA-PK），DNA修复因子具有新的鉴定在RNA代谢和癌症治疗的靶标中的作用，并将使用遗传，细胞生物学和单一的作用分子方法是剖析DNA-PK在淋巴瘤和白血病产生和治疗中的作用的方法。基因组不稳定性是人类癌症的标志。非同源最终连接（NHEJ）是主要的DNA 双链断裂（DSB）修复途径，是生理基因升性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）和大型催化亚基（DNA-PKC）是两个最终过程至关重要的NHEJ因子（例如，发夹开口）和NHEJ期间的结局。 DNA-PKC抑制剂正在I/IIA期癌症临床试验中治疗。在NHEJ期间，KU与DNA末端结合，募集并激活DNA-PKC。 DNA-PKC的损失废除 Artemis核酸内切酶介导的最终过程，而无需废除末端结合。我们表明了激酶死（KD）（DNA-PKCSKD/KD）消除了最终连接，而不会影响最终过程 DNA-PKC的保护作用受其自身的激酶活性调节。 DNA-PKCSKD/KD小鼠中的最终加工被ATM抑制阻塞，表明最终加工需要DNA-PKCS蛋白，而激酶活性 DNA-PKC或体内相关的ATM激酶。 DNA-PKC是DNA-PK的最佳特征底物并且也可以通过ATM磷酸化。携带缺陷磷酸化的小鼠（DNA-PKCS5A/5A）DNA-PKCS 表现出轻度的终端结合缺陷，对ATM抑制敏感。因此，我们建议一旦组装 KU结合的DNA，DNA-PK磷酸化调节最终处理，并最终释放DNA-PKC 许可证终点。此外，我们发现KU还可以指导DNA-PKC在结构化RNA上的组装（例如，rRNA和SNORNA），其中磷酸化有缺陷（DNA-PK5A）或KD DNA-PKC（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。这些发现发现了DNA-PK在哺乳动物中的NHEJ独立作用。和DNA的三分之二 - PKCSKD/KDTRP53 - / - 小鼠屈服于核糖体胁迫诱发髓样白血病，三分之一死于具有IGH-MYC易位的淋巴瘤，突出了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，以抑制淋巴瘤和白血病的起源。到对此进行测试，我们将1）表征和比较RNA与DNA上的KU和DNA-PK动力学； 2）阐明如何耗竭会影响人类细胞中的RNA处理； 3）确定KU80 C末端的生理功能淋巴瘤和白血病发生的结构域和尾巴以及DNA-PKC的募集和稳定。这结果将揭示DNA-PK的RNA和DNA依赖性功能的调节和功能，必需 KU在人类细胞中的作用以及DNA-PK抑制的广泛影响。