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。 尽管多种药理学和免疫学方法用于多模式胶质瘤治疗， 近年来提出的神经胶质瘤表型和基因型空间分布仍然是异质的，因此， 由于治疗耐药性的发展，这是患者结局的最大缺点。 通过永久性细胞融合和临时隧道纳米管（TNT）形成的细胞融合是新颖的， 最近发现的细胞间基因转移和胶质瘤异质性的来源。在我们的补助金中，我们将提供 详细分析了通过细胞融合和TNT进行细胞间基因转移的细胞机制 在患者来源的组织中形成不同胶质瘤亚型，在体外模拟胶质瘤微环境， 以及在小鼠胶质瘤模型体内。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描述：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）在细胞培养后在单细胞水平上的RNA-Seq和WES谱分析（Illumina平台）; 通过流式细胞术技术从解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）分析在细胞相互作用的不同阶段参与基因转移的分子复合物。 细胞特异性Cas9/gRNA指导的基因敲低与靶拯救实验的组合， 细胞融合和TNT形成的基于Cre/荧光的定量报告基因将在体外和体内使用。 体内补充RNA-Seq，WES和蛋白质组学数据，允许识别新靶点并提供 为开发细胞融合和TNT形成的新型药理学抑制剂提供了种子。 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， 细胞毒性和氨基酸剥夺的微环境胶质瘤基因座。我们分析了转录组， 不同分子亚型的五种PDGx细胞系的蛋白质组特征，并证实了 细胞应激反应，即mRNA结合蛋白HuR，是细胞融合和TNT的重要调节因子 在各种应力条件下的地层。TNT形成的关键生物标志物（TNFAIP 2，GJA 1）和 许多内源性融合基因是直接的HuR mRNA靶点，并在神经胶质瘤中以HuR- 依赖的方式。因此，我们建议HuR功能的药理学抑制剂可以作为 由细胞融合和TNT形成引起的细胞间基因转移的抑制因子。在我们的研究中， 我们最近开发并获得专利的HuR二聚化抑制剂（SRI 42127是一种先导化合物） 将在体外和体内胶质瘤异质性的调节中进行评估。高通量全基因组 用人GeCKOv 2 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耐受HuR抑制剂的潜在机制。