BACKGROUND Despite our understanding of the genetic changes that precipitate gliomagenesis, targeted therapy has failed in glioblastoma (GBM) with median survival not significantly improved over the past two decades. Epidermal growth factor receptor (EGFR) alterations, including amplification and activating mutations, are among the most common genetic changes in GBM, occurring in more than half of cases. EGFR is located on Chr. 7, and Chr. 7 gain is one of the earliest events precipitating gliomagenesis. Various EGFR inhibitors, including tyrosine kinase inhibitors, monoclonal antibodies, vaccines, and CAR-T cells have failed in GBM due to intrinsic heterogeneity and receptor tyrosine kinase bypass pathways that mediate therapeutic resistance. New targeted therapeutic approaches to leverage synergistic combinations are desperately needed to improve GBM prognosis. Using the TCGA and other GBM databases, we have previously demonstrated that the presence of PDGFRAamplification in patients with EGFR-amplified GBM carries significantly worse survival. EGFR and PDGFRA co-expression occur in more than one-third of GBM patients. The PDGFRA ligand PDGFA is also located on Chr. 7, and its expression is significantly increased with Chr. 7 gain and EGFR copy number increase. Therefore, Chr. 7 gain inherently leads to co-activation of both EGFR and PDGFRA signaling pathways. MATERIALS AND METHODS We used models of patient-derived glioblastoma cells to test combined inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-alpha in vitro. RESULTS Using patient-derived GBM models with Chr. 7 gain, we found that combined inhibition of both EGFR and PDGFRA using a variety of FDA-approved EGFR-targeted agents (Erlotinib, Gefitinib, Dacomitinib, Neratinib, and Osimertinib) and Crenolanib, respectively, leads to synergistic cytotoxicity in vitro. We found that inhibition of either EGFR or PDGFRA alone led to receptor cross-activation, and EGF and PDGF-AA-induced receptor tyrosine kinase activation was blocked by Neratinib and Crenolanib. Immunoprecipitation experiments and proximity ligation assays demonstrated that combined inhibition prevents EGFR and PDGFRA heterodimerization and pathways of therapeutic resistance. This combined inhibition led to decreased activation of downstream signaling pathways, including phosphatidylinositol 3-kinase and mitogen-activated protein kinase. CONCLUSIONS We show that combined inhibition of EGFR and PDGFRA exerts synergistic cytotoxicity in GBM and prevents resistance pathways that emerge during single-agent targeted therapy against these receptor tyrosine kinases. These pathways are targetable with FDA-approved agents that could be used in patients with GBM with Chr. 7 gain.

中文翻译：

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P13.18 抑制表皮生长因子受体和血小板衍生生长因子受体-α 在胶质母细胞瘤中发挥协同作用

背景尽管我们了解导致胶质瘤发生的遗传变化，但靶向治疗在胶质母细胞瘤 (GBM) 中失败了，在过去的二十年中，中位生存期没有显着改善。表皮生长因子受体 (EGFR) 改变，包括扩增和激活突变，是 GBM 中最常见的遗传变化之一，发生在一半以上的病例中。EGFR 位于 Chr。7，和 Chr。7 增益是促成胶质瘤发生的最早事件之一。由于内在异质性和介导治疗耐药的受体酪氨酸激酶旁路途径，各种 EGFR 抑制剂，包括酪氨酸激酶抑制剂、单克隆抗体、疫苗和 CAR-T 细胞在 GBM 中失败。迫切需要利用协同组合的新靶向治疗方法来改善 GBM 预后。使用 TCGA 和其他 GBM 数据库，我们之前已经证明，在 EGFR 扩增的 GBM 患者中存在 PDGFRA 扩增会显着降低生存率。超过三分之一的 GBM 患者出现 EGFR 和 PDGFRA 共表达。PDGFRA 配体 PDGFA 也位于 Chr。7，它的表达随着 Chr 的增加而显着增加。7增益和EGFR拷贝数增加。因此，Chr。7 增益固有地导致 EGFR 和 PDGFRA 信号通路的共同激活。材料和方法 我们使用患者来源的胶质母细胞瘤细胞模型来测试体外表皮生长因子受体和血小板来源的生长因子受体-α 的联合抑制作用。结果 使用患者衍生的 GBM 模型和 Chr。7 增益，我们发现使用多种 FDA 批准的 EGFR 靶向药物（厄洛替尼、吉非替尼、达克替尼、来拉替尼和奥希替尼）和克雷诺拉尼分别联合抑制 EGFR 和 PDGFRA，导致体外协同细胞毒性。我们发现单独抑制 EGFR 或 PDGFRA 会导致受体交叉激活，而 EGF 和 PDGF-AA 诱导的受体酪氨酸激酶激活被 Neratinib 和 Crenolanib 阻断。免疫沉淀实验和邻近连接测定表明，联合抑制可防止 EGFR 和 PDGFRA 异二聚化和治疗抗性途径。这种联合抑制导致下游信号通路的激活减少，包括磷脂酰肌醇 3-激酶和丝裂原活化蛋白激酶。结论 我们表明，EGFR 和 PDGFRA 的联合抑制在 GBM 中发挥协同细胞毒性作用，并防止在针对这些受体酪氨酸激酶的单药靶向治疗期间出现的耐药途径。这些途径可通过 FDA 批准的药物靶向，可用于患有 Chr 的 GBM 患者。7 增益。