Engineering ERK-specificity for cancer suicide gene therapy

项目总结 DYS通过包括EGFR和MET在内的受体启动的RAS/ERK通路调节的信号是一种 多形性胶质母细胞瘤(GBM)和其他癌症耐药的常见驱动因素。尽管 大力开发这一途径的强大抑制剂，RAS/ERK信号仍然是一个难以捉摸的目标 横跨肿瘤学。这里，我们提出了一种通过ERK在GBM中针对RAS/ERK信令的新方法-- 依赖“自杀基因”的方法。其目的是引入外源基因来驱动选择性 将无毒的前体药物转化为致命物质。HSVtk/GCV(单纯疱疹病毒胸苷 激酶/更昔洛韦)前药系统就是这样一种策略。HSVtk基因与宿主(癌症)的整合 细胞基因组使GCV磷酸化，GCV是2‘-脱氧鸟苷核苷酸的无环类似物，它 在DNA合成过程中与鸟嘌呤竞争。我们最近开发了一种新的策略，其中HSVtk 其表达受ERK活性的调节。具体地说，我们设计了一种表达融合蛋白的病毒载体 HSVtk与转录因子FOS相关抗原1(FRA1)的结构域和核定位 序列。Fra1片段的ERK磷酸化减缓了HSVtk融合的周转，这将成为 在GCV存在的情况下，在足够的表达水平下致死。初步数据表明，GBM细胞 新的自杀基因结构的表达确实可以在ERK中驱动DNA损伤依赖性死亡 依赖活动的方式。在此，我们建议通过两个相辅相成方式推进这项初步工作 明确的目标。在我们的第一个目标中，我们将展示自杀基因产物(称为HSVtk-fire)的能力 选择性杀伤特定类型的GBM肿瘤细胞(与GBM肿瘤中发现的其他细胞类型相比) RAS活性，并测试其与批准的或研究中的疗法合作的能力。我们的第二个目标是， 我们将确定HSVtk-fire是否可以作为治疗GBM小鼠模型的有效方法。这个 第二个目标将是应用对流增强递送来促进病毒载体的递送 将转导带有自杀基因的肿瘤细胞，无论有没有聚焦超声波，这已经被 在初步研究中显示，它可以促进对流增强的大脑传递。最终，这些研究 将提高添加一种强大的新方法的可能性，以在 胶质母细胞瘤和其他癌症。这样的方法是迫切需要的--特别是在新的 尽管有靶向分子的知识，但患者存活率的改善已有多年之久 RAS/ERK信号转导等过程应提供改善患者预后的机会。

PROJECT SUMMARY Dys-regulated signaling through the Ras/ERK pathway, initiated by receptors including EGFR and MET, is a common driver of resistance to therapy in glioblastoma multiforme (GBM), and other cancers. Despite intensive efforts to develop robust inhibitors of this pathway, Ras/ERK signaling remains an elusive target across oncology. Here, we propose a new way to target Ras/ERK signaling in GBM through an ERK- dependent “suicide gene” approach. The aim is to introduce exogenous genes that drive the selective conversion of non-toxic prodrugs to lethal substances. The HSVtk/GCV (Herpes simplex virus thymidine kinase/ganciclovir) prodrug system is one such strategy. Integration of the HSVtk gene into the host (cancer) cell genome enables phosphorylation of GCV, an acyclic analog of the 2’-deoxyguanosine nucleotide, which competes with guanine during DNA synthesis. We recently developed a new strategy in which HSVtk expression is regulated by ERK activity. Specifically, we engineered a viral vector that expresses a fusion of HSVtk with a domain from the transcription factor fos-related antigen 1 (FRA1) and a nuclear localization sequence. ERK phosphorylation of the FRA1 fragment slows the turnover of the HSVtk fusion, which becomes lethal at sufficient expression levels in the presence of GCV. Preliminary data demonstrate that the GBM cell expression of the new suicide gene construct can indeed drive DNA damage-dependent death in an ERK activity-dependent manner. Here, we propose to advance this preliminary work through two complementary specific aims. In our first aim, we will demonstrate the ability of the suicide gene product (termed HSVtk-FIRE) to selectively kill specific GBM tumor cell types (versus other cell types found in GBM tumors) based on high Ras activity and to test its ability to cooperate with approved or investigational therapeutics. In our second aim, we will determine whether HSVtk-FIRE can be used as an effective therapy in mouse models of GBM. The second aim will feature the application of convection-enhanced delivery to promote delivery of viral vectors that will transduce tumor cells with the suicide gene either with or without focused ultrasound, which has been shown in preliminary studies to promote convection-enhanced delivery in the brain. Ultimately, these studies will advance the possibility of adding a powerful new approach for targeting elevated Ras activity in glioblastoma and other cancers. Such approaches are desperately needed—particularly in GBM, where new improvements in patient survival have not occurred in many years despite knowledge of targetable molecular processes such as Ras/ERK signaling that should provide opportunities for improved patient outcomes.