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。 尽管有许多药物和免疫学方法用于多模式脑胶质瘤的治疗 近年来提出的胶质瘤表型和遗传型空间分布仍然是异质性的，因此， 由于治疗耐药性的发展，对患者预后的最大不利因素。 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胶质瘤异质性的来源。在我们的赠款中，我们将提供 详细分析通过细胞融合和TNT进行细胞间基因转移所必需的细胞机制 患者来源组织中不同胶质瘤亚型的形成，体外模拟胶质瘤微环境， 在体内的小鼠胶质瘤模型中也是如此。在肿瘤异质性的背景下细胞融合和TNT的形成 通过以下技术详细描述：i)高分辨率空间细胞转录和组织学图谱 组织切片(Viimum平台)；ii)单细胞水平的RNA-Seq和WES图谱(Illumina平台) 用流式细胞仪技术从分离的组织中进行细胞类型特异性浓缩；III)蛋白质组学(HCP，MASS 光谱分析)分析在细胞相互作用的不同阶段涉及基因转移的分子复合体。 细胞特异性Cas9/gRNA导向的基因敲除与靶向救援实验和 基于Cre/荧光的细胞融合和TNT形成的定量报告将在体外和在 VIVO用于补充RNA-Seq、WES和蛋白质组数据，允许识别新的靶点并提供 用于开发细胞融合和TNT形成的新型药理抑制剂的种子。 我们的初步数据证实了通过细胞融合和TNT形成的水平基因转移 胶质瘤细胞本身和胶质瘤/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胶质瘤基因座。我们分析了转录本和 五个不同分子亚型的PDGx细胞系的蛋白质组学特征，并证实一个中心节点 细胞应激反应，即信使核糖核酸结合蛋白HUR，是细胞融合和TnT的重要调节因子 在多种应力条件下的地层。TNT地层的关键生物标志物(TNFAIP2、GJA1)和 许多内源性融合基因是Hur mRNA的直接靶点，并在Hur-1的胶质瘤中过表达。 依赖的态度。因此，我们认为HUR功能的药物抑制剂可能作为 由细胞融合和TNT形成引起的细胞间基因转移的抑制因子。在我们的赠款中， 最近由我们小组开发并获得专利的HUR二聚化抑制剂(SRI42127是一种先导化合物) 将评估在体外和体内对胶质瘤异质性的调节作用。全基因组的高通量 人GeCKOv2CRISPR全基因组基因敲除文库转导PDGx细胞系的体内筛选 将被用来揭示和补偿细胞融合耐受HUR抑制剂的潜在机制。