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。 尽管采用了多种模式神经胶质瘤治疗的药理和免疫学方法 近年来提出的，神经胶质瘤表型和基因型空间特征仍然是异质的，因此 由于治疗耐药性的发展，代表了患者结局的最大缺点。 通过永久细胞融合和颞隧道纳米管（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进行细胞间基因转移必不可少的细胞机制的详细分析 在患者衍生组织中不同神经胶质瘤亚型的形成，在体外模仿神经胶质瘤微环境， 在体内小鼠神经瘤模型中。在肿瘤异质性的背景下，细胞融合和TNT地层将 通过以下技术详细说明：i）高分辨率的空间细胞转录和组织学分析 组织切片（森林平台）； ii）在单细胞水平上的RNA-Seq和Wes分析（Illumina Platform）之后 通过分离组织的流式细胞仪技术通过流式细胞仪技术的细胞型特异性富集； 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定向基因敲低与目标救援实验和 基于细胞融合和TNT地层的基于定量的CRE/荧光记者将在体外和IN中使用 体内补充RNA-seq，WES和蛋白质组学数据，允许识别新目标并提供 用于开发细胞融合和TNT形成的新药理抑制剂的种子。 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 胶质瘤细胞本身和缺氧，炎症，机械压力的神经胶质瘤/正常宿主细胞， 细胞毒性和氨基酸剥夺的微环境神经胶质瘤基因座。我们分析了转录组和 不同分子亚型的五个PDGX细胞系的蛋白质组学特征，并证实了一个中央节点 细胞应激反应，mRNA结合蛋白HUR，是细胞融合和TNT的必不可少的调节剂 在许多压力条件下的地层。 TNT地层（TNFAIP2，GJA1）和 许多内源性融合基因是直接的HUR mRNA靶标，在Hur-中的神经胶质瘤中过表达 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地层引起的细胞间基因的抑制剂转移。在我们的赠款中， 我们的小组HUR Dimerization抑制剂最近开发和专利（SRI42127是铅化合物） 将在体外和体内的胶质瘤异质性调节中进行评估。全吞吐量全基因组 用人geckov2 CRISPR基因组敲除图书馆转导的PDGX细胞系的体内筛选 将采用用于揭示和补偿细胞融合耐受性对HUR抑制剂的潜在机制。