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.

项目摘要 天然杀手（NK）细胞是先天淋巴样细胞，对于避免病毒感染的宿主防御至关重要 和肿瘤细胞。 NK细胞识别和杀死恶性细胞的能力提供了强大的前提 它们是临床环境中的细胞疗法。而在临床上正在探索基于NK细胞的疗法 试验，持久性和体内功能的问题已被确定为临床的局限性 应用。我们的实验室开创性的细胞因子诱导的记忆样（ML）NK细胞，它们是在短暂后生成的 IL-12，IL-15和IL-18的激活。这克服了常规NK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 急性髓样白血病（AML）的试验，许多患者从这部小说中彻底弥补 干涉。尽管有转化的进步，但对的基本机制知之甚少 ML在NK细胞中重新编程。澄清驱动ML表型的分子程序将具有广泛的 将NK细胞优化为血液恶性肿瘤的一种基于细胞的治疗的意义。 拟议的项目旨在更好地了解ML NK细胞分化的机制，重点 关于EOMES的作用。 EOMES是T-box转录因子（TF），对于NK细胞开发至关重要 造血干细胞已与CD8+ T细胞产生记忆有关。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删除了ML NK细胞分化， 表明EOMES在ML重编程中起关键作用。我们假设使用IL-12/15/18激活 触发常规NK细胞的分子重编程到由Eomes介导的ML NK细胞，以诱导A 独特的转录曲线和固定的表观遗传状态，促进了ML NK的增强功能 细胞通过细胞因子或肿瘤细胞重新刺激。该提案的目标1着重确定记忆 需要EOME的分化阶段。具体来说，我们将评估EOMES的要求 使用CRISPR/CAS9开始（IL-12/15/18激活之前）和分化（IL-12/15/18激活之后） 删除。 AIM 2专注于定义Eomes在Eomes引起的转录和表观遗传景观 ML通过CRISPR/CAS9敲低，单细胞RNASEQ和单核的重新编程的不同阶段 AtacSeq，以及使用切割和运行的ML NK细胞中Eomes调节的基因。在一起，这些 结果将揭示Eomes驱动的ML NK细胞分化的机制，进一步推动了潜在的潜力 ML NK细胞疗法并增强我们对ML NK细胞生物学的理解。