Context-Specific Functional Annotation of EGFR Mutations in Glioblastoma

胶质母细胞瘤中 EGFR 突变的特定背景功能注释

• 批准号: 9394579
• 负责人: Kathleen Kong
• 金额: $ 5.67万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-28 至 2019-06-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9394579
• 关键词: Address; Alleles; Biological; Biological Assay; Brain; CRISPR/Cas technology; Cancer Diagnostics; Catalogs; Cell membrane; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Data Set; Development; Disease; Electroporation; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Erlotinib; Evaluation; Excision; Expression Library; Extracellular Space; Future; Genes; Genetic; Glioblastoma; Goals; Immunoblot Analysis; Individual; Libraries; Malignant Neoplasms; Mediating; Modality; Modeling; Molecular; Molecular Conformation; Mus; Mutagenesis; Mutation; Oncogenic; Operative Surgical Procedures; Pathway interactions; Phosphotransferases; Population; Radiation; Reporting; Research Infrastructure; Resistance; Role; STAT3 gene; Series; Signal Pathway; Signal Transduction; Somatic Mutation; System; Testing; The Cancer Genome Atlas; Therapeutic; Transplantation; Tumor Suppressor Proteins; Tyrosine Kinase Inhibitor; Variant; Work; actionable mutation; base; cancer therapy; cancer type; chemotherapy; clinical translation; data portal; drug sensitivity; genome sequencing; genome-wide; in utero; in vivo; in vivo Model; inhibitor/antagonist; lapatinib; mutant; novel; novel marker; novel therapeutics; receptor function; relating to nervous system; response; small molecule; stem; stem cells; targeted biomarker; therapeutic target; treatment response; tumor; tumorigenesis; validation studies

Project Summary Glioblastoma (GBM) is the most lethal brain malignancy owing to its exceptional resistance to radiation, chemotherapy and surgical resection. While 50% of all GBM cases harbor oncogenic alterations in the gene encoding the Epidermal Growth Factor Receptor (EGFR), it is unclear how the vast majority of those alterations influence EGFR functionality, oncogenic activity and response to available cancer therapeutics. In contrast to EGFR variants found in other types of cancers that involve alterations primarily in the intracellularly- localized kinase domain (KD), the vast majority of EGFR alterations in GBM occur in the ectodomain (EC) extended into the extracellular space. Based on recent studies revealing therapeutic response differences between a small number of EGFR EC and KD variants, we hypothesize that the broader spectrum of oncogenic EGFR EC variants differs functionally from KD mutants with respect to oncogenic signaling and therapeutic vulnerabilities in GBM. The overarching goal of this study is to functionalize the spectrum of EGFR EC and KD aberrations occurring in GBM, as identifying bona fide “driver” mutations and understanding their mechanism of action may inform new cancer diagnostics and therapies. The specific aims of this study are (1) to construct a barcoded EGFR allelic series library, (2) to functionally validate EGFR mutant drivers of GBM, and (3) to determine signaling and therapeutic response differences mediated by oncogenic EGFR EC and KD variants. To address these aims, established high-throughput mutagenesis and molecular barcoding strategies will be leveraged to assemble a library consisting of 64 EGFR variants and controls selected from GBM sequencing data. To determine which mutations contribute to tumorigenesis, EGFR variants will be evaluated for oncogenic activity by employing pooled functional screens using two novel in vivo models that provide the appropriate genetic and biological context of GBM. Specifically, we will combine use of CRISPR-based gene editing of known GBM tumor suppressors with our barcoded EGFR expression libraries for pooled EGFR variant competition assays using 1) an in utero brain electroporation model and 2) a modified neural stem/progenitor cell (NSC) orthotopic transplant model. To investigate mechanistic differences between EGFR EC and KD variants, we will subject NSC cell populations derived from our in vivo studies to immunoblot analysis and drug sensitivity assays to identify differential signaling pathway activation and therapeutic responses by EGFR variants. Successful completion of these studies will not only illuminate the cancer functionality and mechanisms of action of EGFR variants in GBM, but may also uncover novel biomarkers/targets that will immediately inform new therapeutic modalities to treat this fatal disease.

项目摘要 胶质母细胞瘤（GBM）是最致命的脑恶性肿瘤，由于其对辐射的特殊抵抗力， 化疗和手术切除。虽然50%的GBM病例在基因中存在致癌性改变， 编码表皮生长因子受体（EGFR），目前还不清楚绝大多数这些 改变影响EGFR功能、致癌活性和对可用癌症治疗剂的反应。在 与在其他类型的癌症中发现的EGFR变体相反，这些癌症主要涉及细胞内的改变， 局部激酶结构域（KD），GBM中绝大多数EGFR改变发生在胞外域（EC） 延伸到细胞外空间。基于最近的研究显示治疗反应差异 在少数EGFR EC和KD变异体之间，我们假设更广泛的致癌性 EGFR EC变体在致癌信号传导和治疗性免疫应答方面与KD突变体功能不同。 GBM的漏洞本研究的总体目标是功能化EGFR EC和KD谱， GBM中发生的畸变，识别真正的“驱动”突变并了解其机制 可能为新的癌症诊断和治疗提供信息。本研究的具体目的是（1）构建 条形码化的EGFR等位基因系列文库，（2）功能性验证GBM的EGFR突变驱动因子，和（3） 确定致癌EGFR EC和KD变体介导的信号传导和治疗反应差异。 为了解决这些目标，将建立高通量诱变和分子条形码化策略。 用于组装由选自GBM测序的64种EGFR变体和对照组成的文库 数据为了确定哪些突变有助于肿瘤发生，将评估EGFR变体的 通过使用两种新型体内模型进行合并功能筛选来研究致癌活性，这些模型提供了 GBM的适当遗传和生物学背景。具体来说，我们将联合收割机使用基于CRISPR的基因 用我们的条形码化EGFR表达文库编辑已知的GBM肿瘤抑制因子，用于合并EGFR 使用1）子宫内脑电穿孔模型和2）改良的神经电穿孔模型的变体竞争测定 干/祖细胞（NSC）原位移植模型。研究EGFR与 EC和KD变体，我们将对来自我们体内研究的NSC细胞群进行免疫印迹 分析和药物敏感性测定，以鉴定差异信号传导途径活化和治疗 EGFR变异体的反应。这些研究的成功完成不仅将阐明癌症 EGFR变体在GBM中的功能和作用机制，但也可能揭示新的 这些生物标志物/靶标将立即为治疗这种致命疾病的新治疗方式提供信息。