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 自然杀伤(NK)细胞是天然的淋巴样细胞，对宿主抵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进展提供了强有力的前提 作为临床环境中的一种细胞疗法。而以NK细胞为基础的治疗方法正在探索中 试验、持久性和体内功能的问题已被确定为其临床局限性 申请。我们的实验室首创了细胞因子诱导的类记忆(ML)NK细胞，这种细胞在 IL-12、IL-15和IL-18的激活。这克服了传统NK细胞作为一种 细胞疗法。在I期临床中，ML NK细胞表现出增强的功能、细胞毒性和持久性 急性髓系白血病(AML)试验，许多患者从这一新疗法中获得完全缓解 干预。尽管翻译取得了进展，但对其潜在的机制知之甚少。 NK细胞的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细胞作为血液病细胞治疗方法的意义。 建议的项目旨在更好地了解ML NK细胞分化的机制，重点 论伊奥姆斯的作用。Eome是一种T-box转录因子(TF)，对NK细胞的发育至关重要。 造血干细胞，被认为与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，Eome的缺失可以抑制ML NK细胞的分化。 这表明eome在ML重新编程中起着关键作用。我们假设IL-12/15/18的激活 触发由eome介导的常规NK细胞到ML NK细胞的分子重编程，以诱导 独特的转录特征和稳定的表观遗传状态，有助于增强ML NK的功能 细胞在细胞因子或肿瘤细胞的重新刺激下。这项建议的目标1集中在确定记忆 需要EOMES的分化阶段。具体地说，我们将评估EOMS在 使用CRISPR/Cas9启动(在IL-12/15/18激活之前)和分化(在IL-12/15/18激活之后) 删除。目标2集中在定义Eome诱导的转录和表观遗传图景 通过CRISPR/Cas9基因敲除、单细胞RNAseq和单核进行ML重新编程的不同阶段 ATACseq，以及使用Cut&Run定义ML NK细胞中受Eome调控的基因。加在一起，这些 这些结果将揭示Eome诱导ML NK细胞分化的机制，进一步提高 ML NK细胞治疗和增进我们对ML NK细胞生物学的了解。