Crosstalk between EGFR and TEAD Activity Directs Migration in Human Glioblastoma

胶质母细胞瘤(GBM)表现为一个大的、增殖性的核心和弥漫的浸润性边界，后者 导致手术切除不全和不可避免的肿瘤复发。更好地理解 因此，负责基底膜细胞渗透的特定分子通路可能提供新的和潜在的更多 有效的治疗机会。肿瘤细胞的侵袭/迁移生物学是复杂而密切的。 与其微环境有关，包括适应独特的新陈代谢、低氧、运动性、细胞外 基质和神经炎性应激源。这些通路很难在依赖于 关于细胞培养。随着细胞命运的决定在表观遗传控制水平上趋同，一个有吸引力的策略 要研究复杂的迁徙生物学，就是利用表观遗传学来定义 在一个与天然肿瘤细胞状态非常相似的系统中迁移。在这项提议中，我们利用了 我们实验室的表观遗传学专业知识直接研究胶质瘤干细胞群体(GSCs)的细胞命运状态 从他们的肿瘤壁龛中分离出来，没有预先培养的人工制品[37-38，57]。通过独一无二的比较 在新鲜来源的GSC和正常的生发基质祖细胞中，我们能够区分开放染色质 GSCs中与细胞迁移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中最显著的转录因子(TF)基序 与移民有关，在那里，Tead家族成为首选。Tead Tf和他们的合作伙伴- 激活剂YAP/TAZ是河马途径的主要效应因子，并已被研究其致癌作用 主要是在大脑之外发挥调节作用。检测YAP-tead在GBM生长和生长中的功能作用 迁移过程中，我们使用CRISPR/Cas9在患者来源的GSCs中生成了TEAD1基因敲除，并使用了 YAP-TEAD抑制剂维替普芬，用于抑制TEAD活性。使用这两种方法抑制TEAD1活性，我们 检测到体外细胞迁移能力降低，EGFR和PERK表达强烈下调。 EGFR基因敲除同样显示出体外迁移减少，以及TEAD活性降低。 基于此，我们假设TEAD活性调节基底膜的浸润性生长，介导在 至少在一定程度上是通过EGFR监管循环。为了检验这一假设，我们的目标是1)系统地定义 跨GBM亚型开放染色质和下游tead靶基因的tead占有率；2a)解剖 从机制上讲，河马-YAP/TAZ-TEAD和EGFR/RTK-ERK之间的关系定义了 TEAD1在体内外迁移和生长调节中的作用及其与EGFR的协同作用；3)检测 YAP-tead抑制剂的潜在治疗效果，如维替普芬。我们将YAP/TAZ-TEAD设想为 基底膜浸润性生长的重要调节因子，药物抑制其活性可减轻肿瘤 在减轻负担的同时，还可以阻断致癌的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.