Elucidating the role of EOMES in Memory-Like NK cell Differentiation

阐明 EOMES 在记忆样 NK 细胞分化中的作用

• 批准号: 10462945
• 负责人: Jennifer Tran
• 金额: $ 3.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462945
• 关键词: ATAC-seq; Acute Myelocytic Leukemia; B-Lymphocytes; Binding; Biological Assay; CD8-Positive T-Lymphocytes; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Cell Differentiation process; Cell Nucleus; Cell Therapy; Cell division; Cell physiology; Cells; Cellular biology; Chromatin; Clinic; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Cytokine Activation; DNA Binding; Data; Development; Disease; Disease remission; Epigenetic Process; Exhibits; Future; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Haptens; Hematologic Neoplasms; Hematopoietic stem cells; Heterogeneity; Host Defense; Human; In Vitro; Interleukin-12; Interleukin-15; Interleukin-18; Intervention; Knock-out; Ligation; Lymphocyte; Lymphoid Cell; Malignant Neoplasms; Measures; Mediating; Memory; Modification; Molecular; Mus; NK cell therapy; Natural Killer Cells; Patients; Phase; Phase I Clinical Trials; Phenotype; Play; Process; Production; Proteins; Receptor Activation; Refractory; Relapse; Role; Running; Safety; Signal Transduction; T-Lymphocyte; Techniques; Testing; Transcript; Up-Regulation; Virus Diseases; base; cancer cell; clinical application; clinical efficacy; cytokine; cytotoxicity; early phase clinical trial; improved; in vivo; insight; knock-down; multiple omics; neoplastic cell; novel; novel strategies; nuclease; prevent; programs; response; single-cell RNA sequencing; transcription factor; transcriptional reprogramming; transcriptome sequencing; tumor

Project Summary Natural killer (NK) cells are innate lymphoid cells that are crucial for host defense from viral infections and tumor cells. The ability of NK cells to recognize and kill malignant cells provides a strong premise to advance them as a cellular therapy in the clinical setting. While NK cell-based therapies are being explored in clinical trials, issues with persistence and in vivo functionality have been identified as limitations to their clinical application. Our lab pioneered cytokine-induced memory-like (ML) NK cells that are generated after brief activation with IL-12, IL-15, and IL-18. This overcomes many of the shortcomings of conventional NK cells as a cellular therapy. ML NK cells exhibited enhanced functionality, cytotoxicity, and persistence in a phase I clinical trial for acute myeloid leukemia (AML), with many patients achieving complete remissions from this novel intervention. Despite the translational advancement, there is little known about the underlying mechanisms of ML reprogramming in NK cells. Clarifying the molecular programs that drive the ML phenotype will have broad implications for optimizing NK cells as a cellular-based therapy for hematological malignancies. The proposed project aims to better understand the mechanisms of ML NK cell differentiation with a focus on the role of EOMES. EOMES is a T-box transcription factor (TF) that is essential for NK cell development from hematopoietic stem cells and has been implicated in generating memory in CD8+ T cells. Data from our lab demonstrated that EOMES deletion prior to activation with cytokines abrogated ML NK cell differentiation, suggesting that EOMES plays a key role in ML reprogramming. We hypothesize that activation with IL-12/15/18 triggers a molecular reprogramming of conventional NK cells to ML NK cells mediated by EOMES to induce a unique transcriptional profile and poised epigenetic state, which facilitates the enhanced functionality of ML NK cells upon restimulation by cytokines or a tumor cell. Aim 1 of this proposal focuses on determining the memory differentiation phase that requires EOMES. Specifically, we will assess the requirement of EOMES during initiation (prior to IL-12/15/18 activation) and differentiation (after IL-12/15/18 activation) using CRISPR/Cas9 deletion. Aim 2 focuses on defining the transcriptional and epigenetic landscape induced by EOMES in the different phases of ML reprogramming through CRISPR/Cas9 knockdown, single-cell RNAseq, and single-nuclei ATACseq, as well as defining the genes regulated by EOMES in ML NK cells using Cut&Run. Together, these results will reveal mechanisms of ML NK cell differentiation driven by EOMES, further advancing the potential of ML NK cell therapies and enhancing our understanding of ML NK cell biology.

项目概要 自然杀伤 (NK) 细胞是先天性淋巴细胞，对于宿主防御病毒感染至关重要 和肿瘤细胞。 NK细胞识别和杀伤恶性细胞的能力为前进提供了强有力的前提 它们作为临床环境中的细胞疗法。虽然基于 NK 细胞的疗法正在临床探索中 试验中，持久性和体内功能性问题已被确定为其临床的局限性 应用。我们的实验室率先开发了细胞因子诱导的记忆样 (ML) NK 细胞，这些细胞是在短暂的刺激后生成的。 用 IL-12、IL-15 和 IL-18 激活。这克服了传统 NK 细胞作为细胞的许多缺点。 细胞疗法。 ML NK 细胞在 I 期临床中表现出增强的功能、细胞毒性和持久性 急性髓系白血病 (AML) 试验，许多患者从这项新药中获得完全缓解 干涉。尽管转化取得了进展，但人们对其潜在机制知之甚少。 NK 细胞中的 ML 重编程。阐明驱动 ML 表型的分子程序将具有广泛的意义 优化 NK 细胞作为血液恶性肿瘤细胞疗法的意义。 拟议项目旨在更好地了解 ML NK 细胞分化的机制，重点是 关于 EOMES 的作用。 EOMES 是一种 T 盒转录因子 (TF)，对于 NK 细胞发育至关重要 造血干细胞，并与 CD8+ T 细胞的记忆生成有关。来自我们实验室的数据 证明在用细胞因子激活之前删除 EOMES 可消除 ML NK 细胞分化， 表明 EOMES 在机器学习重编程中发挥着关键作用。我们假设 IL-12/15/18 激活 触发由 EOMES 介导的传统 NK 细胞向 ML NK 细胞的分子重编程，从而诱导 独特的转录谱和平衡的表观遗传状态，有助于增强 ML NK 的功能 细胞因子或肿瘤细胞再刺激后的细胞。该提案的目标 1 侧重于确定内存 需要EOMES的分化阶段。具体来说，我们将在期间评估 EOMES 的要求 使用 CRISPR/Cas9 启动（IL-12/15/18 激活之前）和分化（IL-12/15/18 激活之后） 删除。目标 2 侧重于定义 EOMES 诱导的转录和表观遗传景观 通过 CRISPR/Cas9 敲低、单细胞 RNAseq 和单核进行 ML 重编程的不同阶段 ATACseq，以及使用 Cut&Run 定义 ML NK 细胞中 EOMES 调控的基因。在一起，这些 结果将揭示 EOMES 驱动的 ML NK 细胞分化机制，进一步提升 ML NK 细胞分化的潜力 ML NK 细胞疗法并增强我们对 ML NK 细胞生物学的理解。