Investigating PTEN:Ki-67 interaction and its role in DNA damage repair

研究 PTEN:Ki-67 相互作用及其在 DNA 损伤修复中的作用

• 批准号: 10686913
• 负责人: Brandon Marshall Jones
• 金额: $ 4.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10686913
• 关键词: Ablation; Astrocytes; Binding; Binding Proteins; Biological Assay; Biotinylation; CDC2 gene; Cell Death; Cell Nucleus; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA Damage; DNA Repair; Data; Development; Double Strand Break Repair; Engineering; Engraftment; Generations; Glioblastoma; Glioma; Homologous Protein; In Vitro; Ionizing radiation; Knock-in; Knock-in Mouse; Lipids; Malignant - descriptor; Malignant Neoplasms; Mediating; Methods; Missense Mutation; Modality; Modeling; Molecular; Mus; Mutagens; Mutate; Mutation; N-terminal; Nuclear; Oncogenic; PIK3CG gene; PTEN gene; Patients; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Post-Translational Protein Processing; Primary Brain Neoplasms; Publishing; Radiation Tolerance; Radiation therapy; Radiosensitization; Recurrence; Regulation; Resistance; Role; Signal Transduction; Site-Directed Mutagenesis; Streptavidin; Testing; Therapeutic; Transfection; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tyrosine Phosphorylation; Validation; aurora B kinase; chemotherapy; design; epidermal growth factor receptor VIII; experimental study; homologous recombination; in vivo; inhibitor; mimetics; molecular dynamics; mouse model; neoplastic cell; novel; novel therapeutics; overexpression; pharmacologic; prevent; radiation resistance; recruit; response; standard of care; stem-like cell; therapy resistant; tumor

Project Summary Glioblastoma (GBM) is the most aggressive and common malignant primary brain tumor1. Standard of care for GBM patients involves chemotherapy and ionizing radiation (IR) which induce DNA damage to kill tumor cells; however resistance to these treatment modalities commonly develop through various mechanisms2–4. One novel mechanism of radioresistance identified in the Furnari lab is through enhanced homologous recombination (HR) DNA damage response (DDR) mediated by nuclear-localized tyrosine-phosphorylated PTEN (pY240-PTEN) recruited to chromatin through interaction with the PP1 Binding Domain (PP1BD) of Ki-675. PTEN (Phosphatase and Tensin Homolog) is known to play an important tumor-suppressive role and is found to be mutated in approximately 40% of GBMs6. While PTEN cytosolic lipid phosphatase activity inhibition of PI3K signaling has been well established7,8, the function of nuclear PTEN remains less clear. A Y240F-PTEN knock-in mouse model has shown that loss of Y240 phosphorylation results in IR sensitivity5. Preliminary experiments also indicate PTEN:Ki-67 interaction can be disrupted by overexpressed PP1γ, which binds to the RVxF small linear interacting motif (SLiM) located in the Ki-67 PP1BD, and through use of a competitor peptide based on the Ki- 67-Repoman SLiM (KiR-SLiM)5,9. I hypothesize that Ki-67 dependent, pY240-PTEN facilitated DDR is regulated by currently uncharacterized interactions within SLiMs of the Ki-67 PP1BD and that characterization of these interactions will enable development of complex-disrupting peptides capable of radiosensitizing glioma cells. I will firstly identify residues in the Ki-67 KiR-SLiM motif that are essential for pY240-PTEN interaction. This will be accomplished via streptavidin pulldown of N-terminal biotinylated peptides designed based on the KiR-SLiM. Orthogonally, Ki-67 minigene constructs harboring mutation of the identified PP1BD residues will be utilized in GBM models to examine their effects on PTEN:Ki-67 interaction, as well as DDR, HR, chromatin accessibility, and colony formation efficiency. Competitor peptides will be designed based on candidate Ki-67 residues and evaluated for their ability to disrupt PTEN:Ki-67 interaction and radiosensitize glioma cells. Secondly, regulation of pY240-PTEN:Ki-67 interaction by posttranslational modification of Ki-67 SLiMs by aurora B kinase and cyclin dependent kinase 1 will be investigated through site directed mutagenesis and pharmacological inhibition. Discoveries regarding SLiM regulation will be incorporated into competitor peptides to optimize specific disruption of the pY240-PTEN:Ki-67 complex. Lastly, mutations determined to have the greatest impact on pY240-PTEN:Ki-67 interactions will be CRISPR edited into oncogenic mouse astrocytes with WT versus knock-in Y240F PTEN and the effects of these mutations will be investigated in vitro and in vivo5. Overall, this project aims to characterize the regulation and critical molecular interactions between pY240-PTEN and Ki-67, culminating in strategies that will disrupt these interactions to enhance the efficacy of radiotherapy.

项目摘要 胶质母细胞瘤（GBM）是最具侵袭性和最常见的恶性原发性脑肿瘤1。标准治疗 GBM患者涉及化疗和电离辐射（IR），其诱导DNA损伤以杀死肿瘤细胞; 然而，对这些治疗方式的耐药性通常通过各种机制2 -4产生。一个新颖 Furnari实验室确定的辐射抗性机制是通过增强的同源重组（HR） 核定位酪氨酸磷酸化的PTEN（pY 240-PTEN）介导的DNA损伤反应（DDR） 通过与Ki-675的PP 1结合结构域（PP 1BD）相互作用募集到染色质。PTEN（磷酸酶 和张力蛋白同源物）已知发挥重要的肿瘤抑制作用，并且发现在 约40%的GBMs 6。而PTEN细胞质脂质磷酸酶活性抑制PI 3 K信号传导， 虽然已经很好地建立了7，8，但核PTEN的功能仍然不太清楚。Y240 F-PTEN基因敲入小鼠模型的建立 Y240磷酸化的丧失导致IR敏感性5。初步实验还表明 PTEN：Ki-67相互作用可被过表达的PP 1 γ破坏，PP 1 γ结合到RpGF 16小线性区。 相互作用基序（SLiM）位于Ki-67 PP 1BD，并通过使用基于Ki-67 PP 1BD的竞争肽， 67-Repoman SLiM（KiR-SLiM）5，9.我假设Ki-67依赖性，pY 240-PTEN促进的DDR， 受Ki-67 PP 1BD的SLiM内目前未表征的相互作用调节， 对这些相互作用的表征将使得能够开发能够破坏复合物的肽， 放射增敏神经胶质瘤细胞。我将首先鉴定Ki-67 KiR-SLiM基序中的残基，这些残基对于Ki-67 KiR-SLiM基序的表达是必需的。 pY 240-PTEN相互作用。这将通过N-末端生物素化肽的链霉亲和素下拉来实现 基于KiR-SLiM设计。从分子水平上看，Ki-67小基因构建体携带所鉴定的 PP 1BD残基将用于GBM模型中，以检查其对PTEN：Ki-67相互作用的影响，以及 DDR、HR、染色质可及性和集落形成效率。竞争肽将基于 对候选Ki-67残基进行修饰，并评估其破坏PTEN：Ki-67相互作用和放射增敏的能力 胶质瘤细胞第二，通过Ki-67的翻译后修饰调节pY 240-PTEN：Ki-67相互作用 将通过定点突变研究极光B激酶和细胞周期蛋白依赖性激酶1的SLiM 和药理学抑制。有关SLiM法规的发现将纳入竞争对手 肽以优化pY 240-PTEN：Ki-67复合物的特异性破坏。最后，突变决定了 对pY 240-PTEN：Ki-67相互作用的最大影响将是CRISPR编辑到致癌小鼠星形胶质细胞中 用WT与敲入Y240 F PTEN比较，并将在体外和体内研究这些突变的影响5。 总之，本项目旨在表征pY 240-PTEN之间的调节和关键分子相互作用 和Ki-67，最终的战略，将破坏这些相互作用，以提高放射治疗的疗效。