ELAVL1 role in glioblastoma heterogeneity through intercellular gene transfer mediated by cell fusion and tunneling membrane nanotube formation

ELAVL1通过细胞融合和隧道膜纳米管形成介导的细胞间基因转移在胶质母细胞瘤异质性中的作用

• 批准号: 10658226
• 负责人: Natalia Filippova
• 金额: $ 33.97万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-08 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10658226
• 关键词: Amino Acids; Architecture; Autophagocytosis; Back; Binding Proteins; Biological; Biological Markers; Brain; Cell Communication; Cell Line; Cell Survival; Cell fusion; Cells; Cellular Assay; Cellular Stress; Cerebrum; Clustered Regularly Interspaced Short Palindromic Repeats; Compensation; Complement; Complex; Cytoplasm; Cytoskeleton; Data; Data Analyses; Development; Dimerization; Disadvantaged; Dissection; Dissociation; Drug resistance; ELAV protein; Event; Family; Female; Flow Cytometry; Fluorescence; Gene Expression Profiling; Gene Transfer; Genes; Genetic Transcription; Genomic Instability; Genomics; Genotype; Glioblastoma; Glioma; Grant; Guide RNA; Heterogeneity; Histologic; Horizontal Gene Transfer; HuR protein; Human; Human Genome; Hypoxia; Immunocompetent; Immunocompromised Host; Immunologics; In Vitro; Inflammatory; Knock-out; Lead; Legal patent; Libraries; Mass Spectrum Analysis; Mechanical Stress; Mechanics; Mediating; Membrane; Membrane Fusion; Messenger RNA; Metabolic; Microfluidics; Mitochondria; Modality; Modeling; Molecular; Molecular Analysis; Mus; Nanotubes; Normal Cell; Nutrient; Organoids; Outcome; Patient-Focused Outcomes; Patients; Phenotype; Phosphatidylserines; Process; Proteins; Proteomics; RNA-Binding Proteins; Regulation; Reporter; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Protein; Slice; Source; Stress; Surface; Techniques; Tissues; Tube; Up-Regulation; cell mediated gene transfer; cell transformation; cell type; cytotoxic; experimental study; extracellular vesicles; genome wide screen; genome-wide; genome-wide analysis; in vivo; in vivo Model; inhibitor; knock-down; knockout gene; male; molecular subtypes; multimodality; neoplastic; novel; novel marker; overexpression; pharmacologic; prognostic signature; protein complex; proteomic signature; receptor; screening; therapy development; therapy resistant; transcriptome; transcriptome sequencing; transcriptomics; tumor; tumor heterogeneity

Project Summary. Despite the numerous pharmacological and immunological approaches for multimodal glioma treatments proposed in recent years, glioma phenotypic and genotypic spatial profiles remain heterogeneous and, therefore, represent the biggest disadvantage for patient outcomes due to the development of treatment resistance. Cell fusions through permanent cell-fusion and temporal tunneling nanotube (TNT) formations are novel, recently discovered sources of intercellular gene transfers and glioma heterogeneity. In our grant, we will provide a detailed analysis of cellular mechanisms essential for intercellular gene transfer via cell fusion and TNT formations for different glioma subtypes in patient-derived tissue, mimicked glioma microenvironment in vitro, and in mouse glioma models in vivo. Cell fusion and TNT formations in the context of tumor heterogeneity will be detailed by following techniques: i) the high-resolution spatial cell transcriptional and histological profiling in tissue slices (Visium platform); ii) the RNA-Seq and WES profiling (Illumina platform) on a single-cell level after cell-type-specific enrichment by flow cytometry technique from dissociated tissue; iii) the proteomics (HCP, mass spectrometry) analysis of molecular complexes involved in gene transfer at different stages of cell interaction. Cell-specific Cas9/gRNA- directed gene knockdown in combination with the target rescue experiments and quantitative Cre/fluorescence-based reporters of cell fusion and TNT formations will be utilized in vitro and in vivo to complement RNA-Seq, WES, and proteomics data, allowing identification of new targets and providing seeds for the development of novel pharmacological inhibitors of cell fusion and TNT formations. Our preliminary data identified horizontal gene transfer via cell fusion and TNT formations between glioma cells themselves and glioma/normal host cells in the hypoxic, inflammatory, mechanically stressed, cytotoxic, and amino acid deprived microenvironmental glioma loci. We analyzed the transcriptomic and proteomic signature of five PDGx cell lines of different molecular subtypes and confirmed that a central node of the cellular stress response, the mRNA-binding protein HuR, is an essential regulator of cell fusion and TNT formations in numerous stress conditions. The key biomarkers of TNT formations (TNFAIP2, GJA1) and numerous endogenous fusogenes are direct HuR mRNA targets and overexpressed in gliomas in a HuR- dependent manner. Therefore, we propose that pharmacological inhibitors of HuR function may serve as suppressors of intercellular gene transfers evoked by cell fusion and TNT formations. In our grant, the impact of the recently developed and patented by our group inhibitors of HuR dimerization (SRI42127 is a lead compound) will be assessed in the regulation of glioma heterogeneity in vitro and in vivo. The high throughput genome-wide in vivo screening of PDGx cell lines transduced with a Human GeCKOv2 CRISPR genome-wide knockout library will be employed to reveal and compensate potential mechanisms of cell fusion tolerance to the HuR inhibitors.

项目摘要。 尽管有多种用于多模式神经胶质瘤治疗的药理学和免疫学方法 近年来提出，神经胶质瘤的表型和基因型空间分布仍然存在异质性，因此， 由于治疗耐药性的发展，这是对患者结果的最大不利。 通过永久细胞融合和时间隧道纳米管（TNT）形成的细胞融合是新颖的， 最近发现了细胞间基因转移和神经胶质瘤异质性的来源。在我们的赠款中，我们将提供 通过细胞融合和 TNT 详细分析细胞间基因转移所必需的细胞机制 患者来源组织中不同神经胶质瘤亚型的形成，模拟体外神经胶质瘤微环境， 以及小鼠体内神经胶质瘤模型。肿瘤异质性背景下的细胞融合和 TNT 形成将 通过以下技术进行详细说明：i）高分辨率空间细胞转录和组织学分析 组织切片（Visium 平台）； ii) 单细胞水平上的 RNA-Seq 和 WES 分析（Illumina 平台） 通过流式细胞术技术从分离的组织中进行细胞类型特异性富集； iii) 蛋白质组学（HCP、质量 光谱法）分析细胞相互作用不同阶段参与基因转移的分子复合物。 细胞特异性 Cas9/gRNA 定向基因敲除结合靶标拯救实验 基于 Cre/荧光的细胞融合和 TNT 形成的定量报告将在体外和体内使用 vivo 补充 RNA-Seq、WES 和蛋白质组学数据，从而识别新靶点并提供 开发细胞融合和 TNT 形成的新型药理学抑制剂的种子。 我们的初步数据确定了通过细胞融合和 TNT 形成的水平基因转移 神经胶质瘤细胞本身和神经胶质瘤/正常宿主细胞在缺氧、炎症、机械应激、 细胞毒性和氨基酸缺失的微环境神经胶质瘤位点。我们分析了转录组和 五个不同分子亚型的 PDGx 细胞系的蛋白质组学特征，并证实了 细胞应激反应，mRNA 结合蛋白 HuR，是细胞融合和 TNT 的重要调节因子 在许多应力条件下形成。 TNT 形成的关键生物标志物（TNFAIP2、GJA1）和 许多内源性融合基因是 HuR mRNA 的直接靶点，并在 HuR 的神经胶质瘤中过度表达。 依赖方式。因此，我们建议 HuR 功能的药理学抑制剂可以作为 细胞融合和 TNT 形成引起的细胞间基因转移的抑制因子。在我们的资助中，影响 我们集团最近开发并获得专利的 HuR 二聚抑制剂（SRI42127 是先导化合物） 将评估体外和体内神经胶质瘤异质性的调节。高通量全基因组 使用人类 GeCKOv2 CRISPR 全基因组敲除文库转导的 PDGx 细胞系的体内筛选 将用于揭示和补偿细胞融合对 HuR 抑制剂耐受的潜在机制。