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

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脑肿瘤。护理的标准 GBM患者包括化疗和电离辐射(IR)，这会导致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是通过各种机制2-4产生的。 在Furnari实验室中发现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PTEN(pY240-PTEN)介导的DNA损伤反应 通过与Ki-675的PP1结合结构域(PP1BD)相互作用而被招募到染色质。PTEN(磷酸酶) 和Tensin Homolog)已知具有重要的肿瘤抑制作用，并被发现在 大约40%的GBM 6。而PTEN细胞内脂磷酸酶活性抑制PI3K信号转导 核PTEN的功能已经被很好地建立了7，8，但仍然不清楚。Y240F-PTEN基因敲除小鼠模型的建立 已经表明，Y240磷酸化的缺失会导致IR敏感性5。初步实验还表明 PTEN：Ki-67的相互作用可以被过度表达的pp1γ所破坏，它与RVxF小分子线性结合 定位于Ki-67 PP1BD的相互作用基序(SLIM)，并通过使用基于Ki-67 PP1BD的竞争多肽。 67-Repoman SLIM(KIR-SLIM)5，9.我假设依赖Ki-67，pY240-PTEN促进的DDR是 受目前尚不清楚的Ki-67 PP1BD分子内相互作用的调节 这些相互作用的表征将使开发能够破坏复杂结构的多肽成为可能 对辐射敏感的胶质瘤细胞。我将首先确定Ki-67 Kir-SLIM基序中的残基，它们对 PY240-PTEN相互作用。这将通过N端生物素化多肽的链霉亲和素下拉来实现 基于KIR-SLIM设计。正交性地构建含有已鉴定突变的Ki-67微型基因 PP1BD残基将用于GBM模型，以检查它们对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。竞争对手的多肽将基于 候选Ki-67残基及其破坏PTEN：Ki-67相互作用和放射增敏能力的评价 胶质瘤细胞。第二，Ki-67翻译后修饰对pY240-PTEN：Ki-67相互作用的调节 极光B激酶和细胞周期蛋白依赖性激酶1的瘦身将通过定点突变进行研究 和药理抑制作用。有关精简监管的发现将纳入竞争对手 优化pY240-PTEN：Ki-67复合体的特异性破坏的多肽。最后，被确定为具有 对pY240-PTEN的最大影响：Ki-67相互作用将被CRISPR编辑成致癌的小鼠星形胶质细胞 与WT和敲入Y240F PTEN相比，这些突变的影响将在体外和体内进行研究。 总体而言，本项目旨在表征pY240-PTEN之间的调控和关键的分子相互作用 和Ki-67，最终的策略将破坏这些相互作用，以提高放射治疗的疗效。

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.