Crosstalk between EGFR and TEAD Activity Directs Migration in Human Glioblastoma

胶质母细胞瘤（GBM）肿瘤表现为一个大的，增殖的核心和弥漫性浸润的边界，后者 导致不完全的手术切除和不可避免的肿瘤复发。更好地理解 因此，负责GBM中细胞浸润的特定分子途径可以提供新的和潜在的更多的 有效的治疗机会。肿瘤细胞浸润/迁移的生物学是复杂且密切相关的 与其微环境，包括适应独特的代谢，缺氧，运动，细胞外 基质和神经炎性应激源。这些途径很难在依赖于神经胶质瘤模型的神经胶质瘤模型中重现。 在细胞培养上。随着细胞命运决定在表观遗传控制水平上的收敛，一个有吸引力的策略 研究迁移的复杂生物学是利用表观遗传学来定义转录调节因子， 在一个系统中的迁移，非常类似于天然肿瘤细胞的状态。在本提案中，我们利用 我们实验室的表观遗传学专业知识，研究胶质瘤干细胞群（GSC）中的细胞命运状态，直接 从其肿瘤小生境中分离，无预培养伪影[37- 38，57]。通过独特的比较 在新鲜衍生的GSC和正常的骨髓基质祖细胞中开放染色质，我们能够区分 GSC中与细胞迁移特异相关的转录可及区域（最终修订版）。这 使我们能够推断出肿瘤特异性调控区域内最显著的转录因子（TF）基序 与移民有关，TEAD家族成为首选。TEAD TF，沿着他们的合作伙伴， 激活剂雅普/TAZ是Hippo途径的主要效应物，并且已经对其致癌性进行了研究。 主要在大脑外部发挥调节作用。为了测试YAP-TEAD在GBM生长中的功能作用， 迁移，我们使用CRISPR/Cas9在患者来源的GSC中产生TEAD 1敲除，并采用 YAP-TEAD抑制剂维替泊芬抑制TEAD活性。使用这两种方法抑制TEAD 1活性，我们 检测到体外细胞迁移能力降低和EGFR和pERK表达的稳健下调。 EGFR敲除类似地显示出体外迁移减少以及TEAD活性降低。 基于此，我们假设TEAD活性调节GBM的浸润性生长，介导于 至少部分通过EGFR调节环。为了验证这一假设，我们的目标是：1）系统地定义 跨GBM亚型的开放染色质和下游TEAD靶基因处的TEAD占据率; 2a）解剖 Hippo-YAP/TAZ-TEAD和EGFR/RTK-ERK之间的关系; 2b）定义Hippo-YAP/TAZ-TEAD和EGFR/RTK-ERK之间的作用; TEAD 1在体外和体内/离体调节迁移和生长中的作用，以及其与EGFR的协同作用;以及3）测试TEAD 1在体外和体内/离体调节迁移和生长中的作用。 YAP-TEAD抑制剂，如维替泊芬的潜在治疗效果。我们设想雅普/塔兹-泰德作为一个 GBM中浸润性生长的重要调节因子，其活性的药理学抑制可使肿瘤恶化 同时也阻断致癌RTK效应，从而为GBM患者提供新的治疗选择。

Glioblastoma (GBM) tumors manifest with a large, proliferative core and a diffusely infiltrative border, the latter contributing to incomplete surgical resection and inevitable tumor recurrence. A better understanding of the specific molecular pathways responsible for cell infiltration in GBM may thus provide novel and potentially more effective opportunities for therapy. The biology of tumor cell infiltration/migration is complex and intimately linked to its microenvironment, including adaptations for unique metabolic, hypoxic, motility, extracellular matrix, and neuroinflammatory stressors. The pathways are difficult to recapitulate in glioma models that rely on cell culturing. As cell fate decisions converge on the level of epigenetic control, one attractive strategy to study the intricate biology of migration is to use epigenetics to define the transcriptional regulators of migration in a system that closely resembles the native tumor cell state. In this proposal, we take advantage of our lab's epigenetic expertise to study cell fate states in glioma stem cell populations (GSCs), directly isolated from their tumor niche without pre-culture artifact [37-38,57]. Through the unique comparison of open chromatin in freshly derived GSCs and normal germinal matrix progenitors, we were able to distinguish the transcriptionally accessible regions in GSCs that specifically relate to cell migration (in final revisions). This allowed us to infer the most salient transcription factor (TF) motifs within tumor-specific regulatory regions linked to migration, where the TEAD family emerged as a top candidate. The TEAD TFs, along with their co- activators YAP/TAZ, are the main effectors of the Hippo pathway, and have been studied for their oncogenic regulatory role predominantly outside of the brain. To test the functional role of YAP-TEAD in GBM growth and migration, we generated TEAD1 knockout in patient-derived GSCs using CRISPR/Cas9 and employed the YAP-TEAD inhibitor, Verteporfin, to inhibit TEAD activity. Using both methods to inhibit TEAD1 activity, we detected reduced capacity for cell migration in vitro and robust downregulation of EGFR and pERK expression. EGFR knockout similarly displayed reduced migration in vitro, as well as decreased TEAD activity. Based on this, here we hypothesize that TEAD activity regulates infiltrative growth in GBM, mediated at least partially through an EGFR regulatory loop. To test this hypothesis, we aim to 1) systematically define TEAD occupancy at open chromatin and downstream TEAD-target genes across GBM subtypes; 2a) dissect mechanistically the relationship between Hippo-YAP/TAZ-TEAD and EGFR/RTK-ERK; 2b) define the role of TEAD1 in regulating migration and growth in vitro and in/ex vivo, and its synergy with EGFR; and 3) test the potential therapeutic efficacy of YAP-TEAD inhibitors, such as Verteporfin. We envision YAP/TAZ-TEAD as an important regulator of infiltrative growth in GBM, where pharmacologic inhibition of its activity alleviates tumor burden while also blocking oncogenic RTK effects, thus offering new therapeutic options for patients with GBM.