Investigating the role of Cdk5 and p35 in natural killer cell cytotoxicity

研究 Cdk5 和 p35 在自然杀伤细胞细胞毒性中的作用

基本信息

批准号：
10701749
负责人：
Derek Perseus Wong
金额：
$ 5.27万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
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：
2022-09-08 至 2025-09-07
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10701749
关键词：
Activated Natural Killer Cell Adoptive Cell Transfers Agreement Allogenic Antibodies Antigen-Presenting Cells B-Cell Acute Lymphoblastic Leukemia Binding Biological Assay Cancer Patient Cancer cell line Cell Cycle Cell Line Cell-Mediated Cytolysis Cells Cyclin-Dependent Kinase 5 Cyclin-Dependent Kinase Inhibitor Cyclin-Dependent Kinases Cyclins Cytoplasmic Granules Data Dose Down-Regulation Enzymes Epithelium Exhibits Exposure to FCGR3B gene Face Family Functional disorder Genetic Goals Granzyme Hematologic Neoplasms Histone H1 Human Immune Immune system Immunologic Surveillance Immunoprecipitation In Vitro Innate Immune System Interferon Type II Interleukin-2 Knock-out Lymphocyte Lytic Malignant Neoplasms Measures Mediating Mediator Mesenchymal Molecular Mus NK cell therapy Natural Killer Cell Immunotherapy Natural Killer Cell toxicity Natural Killer Cells Neurons Nuclear Pathway interactions Patients Phenotype Phosphorylation Phosphotransferases Play Proline Protein-Serine-Threonine Kinases Proteins Receptor Inhibition Reporting Research Risk Role Serine Signal Pathway Signal Transduction Signaling Protein Stains T cell therapy T-Cell Activation T-Lymphocyte T-bet protein TGF Beta Signaling Pathway TNF gene Testing Therapeutic Threonine Time Transforming Growth Factor beta Transforming Growth Factor beta Receptors Tumor Burden Up-Regulation Viral anti-cancer antibody-dependent cell cytotoxicity cancer cell cancer immunotherapy cancer therapy cell type chromatin immunoprecipitation cytokine cytotoxic cytotoxicity differential expression efficacy evaluation exhaustion experimental study graft vs host disease immune function improved in vivo inhibiting antibody inhibitor knock-down lentivirally transduced malignant breast neoplasm member mouse model mutant neoplastic cell overexpression patient derived xenograft model perforin promoter protein expression receptor roscovitine small hairpin RNA tool transcription factor transcriptome sequencing tumor

项目摘要

PROJECT SUMMARY/ABSTRACT Natural killer (NK) cells are cytotoxic lymphocytes with important immune functions in killing virally infected cells and cancer cells. NK cells have been explored for cancer immunotherapy and have advantages over T cell- based therapies. However, their cytotoxicity and tumor immunosurveillance functions are often dysfunctional in cancer patients, in large part due to elevated levels of TGF-β, a potent immunosuppressive cytokine. Cyclin- dependent kinase 5 (Cdk5) is a Cdk family proline-directed serine/threonine kinase. Unlike other Cdk members, its kinase activity is primarily dependent on binding of the coactivator p35 or p39, and it has unclear cell cycle roles. Cdk5 was thought to primarily function in neuronal cells, but recent research has discovered new roles for Cdk5 and p35 in other cell types, including cancer and immune cells. For the first time, we have discovered that Cdk5 and p35 protein are both expressed in NK cells and appear to play an important role in regulating NK cell cytotoxicity. Additionally, TGF-β appears to induce p35 expression in NK cells in a dose-dependent manner. Based on our preliminary data, we hypothesize that Cdk5/p35 kinase activity negatively regulates NK cell cytotoxicity and is a key mediator of TGF-β-induced NK cell dysfunction, and we also hypothesize that Cdk5/p35 inhibition can be utilized to enhance NK cell immunotherapy. First, we will explore how the Cdk5/p35 and TGF- β signaling pathways overlap in NK cells. Using genetic tools to knock down p35, as well as the selective Cdk5 inhibitor roscovitine, we will determine whether Cdk5-inhibited NK cells can mitigate the various phenotypic changes caused by TGF-β treatment. We will measure any changes in the expression of NK cell activating/inhibitory receptors, lytic granule cytotoxic enzymes, and cytokine release. We will also determine how TGF-β induces p35 expression in NK cells, then investigate the molecular mechanism of how Cdk5/p35 activity regulates NK cytotoxicity. Whole transcriptome sequencing of p35 knockdown NK cells will be used to reveal differentially expressed pathways downstream of Cdk5 kinase signaling. We also wish to explore the therapeutic potential of Cdk5/p35 inhibition in enhancing NK cell immunotherapy. Using in vitro cytotoxicity assays against cancer cell lines, we will determine whether p35 knockdown will enable NK cells to resist TGF-β-induced suppression of cytotoxicity. Similarly, using established patient-derived xenograft mouse models, we will test whether p35 knockdown NK cells are able to enhance NK cell adoptive therapy against patient-derived B cell acute lymphoblastic leukemia (B-ALL), which is known to cause NK dysfunction through elevated TGF-β secretion. Discoveries from this project would advance our basic understanding of the signaling pathways that regulate NK cytotoxicity and mediate NK dysfunction, potentially leading to improved NK cell-based cancer immunotherapies.

项目概要/摘要自然杀伤 (NK) 细胞是细胞毒性淋巴细胞，在杀死病毒感染方面具有重要的免疫功能 NK细胞已被探索用于癌症免疫治疗，并且比T细胞具有优势。然而，它们的细胞毒性和肿瘤免疫监视功能通常功能失调。癌症患者，很大程度上是由于 TGF-β（一种有效的免疫抑制细胞因子）水平升高。依赖性激酶 5 (Cdk5) 是一种 Cdk 家族脯氨酸定向丝氨酸/苏氨酸激酶，与其他 Cdk 成员不同。其激酶活性主要依赖于辅激活因子p35或p39的结合，并且其细胞周期不清楚人们认为 Cdk5 主要在神经元细胞中发挥作用，但最近的研究发现了新的作用。我们首次发现其他细胞类型（包括癌症和免疫细胞）中的 Cdk5 和 p35。 Cdk5 和 p35 蛋白均在 NK 细胞中表达，似乎在调节 NK 细胞中发挥重要作用此外，TGF-β 似乎以剂量依赖性方式诱导 NK 细胞中的 p35 表达。根据我们的初步数据，我们勇敢地认为 Cdk5/p35 激酶活性对 NK 细胞有负调节作用细胞毒性，是 TGF-β 诱导的 NK 细胞功能障碍的关键介质，我们还捕获了 Cdk5/p35 首先，我们将探讨 Cdk5/p35 和 TGF-β 的抑制作用如何增强 NK 细胞免疫治疗。 NK 细胞中的 β 信号通路重叠。使用遗传工具敲除 p35 以及选择性 Cdk5。抑制剂roscovitine，我们将确定Cdk5抑制的NK细胞是否可以减轻各种表型我们将测量 NK 细胞表达的任何变化。我们还将确定如何激活/抑制受体、裂解颗粒细胞毒性酶和细胞因子释放。 TGF-β诱导NK细胞中p35表达，然后研究Cdk5/p35活性的分子机制 p35 敲低 NK 细胞的全转录组测序将用于揭示调节 NK 细胞毒性。我们还希望探索 Cdk5 激酶信号下游的差异表达途径。 Cdk5/p35 抑制在增强 NK 细胞免疫治疗中的潜力。癌细胞系，我们将确定 p35 敲低是否会使 NK 细胞抵抗 TGF-β 诱导的同样，我们将使用已建立的患者来源的异种移植小鼠模型进行测试。 p35 敲低 NK 细胞是否能够增强针对患者来源 B 细胞的 NK 细胞过继治疗急性淋巴细胞白血病 (B-ALL)，已知会通过 TGF-β 升高导致 NK 功能障碍该项目的发现将增进我们对信号通路的基本了解。调节 NK 细胞毒性并介导 NK 功能障碍，有可能改善基于 NK 细胞的癌症免疫疗法。