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

• 批准号: 10535188
• 负责人: Derek Perseus Wong
• 金额: $ 5.18万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 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/10535188
• 关键词: 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; Epithelial; Exhibits; Exposure to; FCGR3B gene; Face; Family; Functional disorder; GTP-Binding Protein alpha Subunits, Gs; Genetic; Goals; Granzyme; Hematologic Neoplasms; Histone H1; Human; Immune; Immune system; Immunologic Surveillance; Immunoprecipitation; Immunotherapy; In Vitro; Innate Immune System; Interferon Type II; Interleukin-2; Knock-out; Lymphocyte; Lytic; Malignant Neoplasms; Measures; Mediating; Mediator of activation protein; Mesenchymal; Molecular; Mus; NK cell therapy; 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; base; cancer cell; cancer immunotherapy; cancer therapy; cell type; chromatin immunoprecipitation; cytokine; cytotoxic; cytotoxicity; differential expression; exhaustion; experimental study; graft vs host disease; immune function; improved; in vivo; 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

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.

项目总结/摘要 自然杀伤（NK）细胞是一类具有杀伤病毒感染的细胞毒性淋巴细胞，具有重要的免疫功能 细胞和癌细胞。NK细胞已被探索用于癌症免疫治疗，并具有优于T细胞的优势。 基础疗法。然而，它们的细胞毒性和肿瘤免疫监视功能在肿瘤中通常是功能失调的。 在癌症患者中，这在很大程度上是由于TGF-β（一种有效的免疫抑制细胞因子）水平升高。细胞周期蛋白 依赖性激酶5（Cdk 5）是Cdk家族脯氨酸导向的丝氨酸/苏氨酸激酶。与其他CDK成员不同， 其激酶活性主要依赖于与辅激活因子p35或p39的结合，细胞周期不清楚 角色cdk 5被认为主要在神经元细胞中起作用，但最近的研究发现了cdk 5的新作用。 cdk 5和p35在其他细胞类型，包括癌症和免疫细胞。我们首次发现 Cdk 5和p35蛋白均在NK细胞中表达，并在NK细胞的增殖和分化过程中起重要作用。 细胞毒此外，TGF-β似乎以剂量依赖性方式诱导NK细胞中的p35表达。 基于我们的初步数据，我们假设Cdk 5/p35激酶活性负调控NK细胞， 细胞毒性，是TGF-β诱导的NK细胞功能障碍的关键介质，我们还假设Cdk 5/p35 抑制可用于增强NK细胞免疫治疗。首先，我们将探讨Cdk 5/p35和TGF-β 1如何在细胞内表达。 β信号传导途径在NK细胞中重叠。使用遗传工具敲除p35以及选择性Cdk 5 我们将确定Cdk 5抑制的NK细胞是否可以减轻各种表型 TGF-β治疗引起的变化。我们将测量NK细胞表达的任何变化， 活化/抑制受体、溶解颗粒细胞毒性酶和细胞因子释放。我们还将确定如何 TGF-β诱导NK细胞p35表达，探讨Cdk 5/p35活性的分子机制 调节NK细胞毒性。p35敲低NK细胞的全转录组测序将用于揭示 Cdk 5激酶信号传导下游的差异表达途径。我们还希望探索治疗 Cdk 5/p35抑制在增强NK细胞免疫治疗中的潜力。使用体外细胞毒性测定， 在肿瘤细胞系中，我们将确定p35敲低是否能够使NK细胞抵抗TGF-β诱导的 抑制细胞毒性。同样，使用已建立的患者来源的异种移植小鼠模型，我们将测试 p35敲低的NK细胞是否能够增强NK细胞对患者来源的B细胞的过继治疗 急性淋巴细胞白血病（B-ALL），已知通过升高TGF-β导致NK功能障碍 分泌物该项目的发现将促进我们对信号通路的基本理解， 调节NK细胞毒性并介导NK功能障碍，可能导致改善基于NK细胞的癌症 免疫疗法