Investigating Alpha-secretase inhibitors as Notch inhibitors & anti-glioma agents

描述（由申请人提供）：尽管在过去的二十年里，在治疗其他癌症方面取得了显著的进展，但脑肿瘤仍然是一个令人生畏的治疗挑战。这些肿瘤对放射和化疗的标准治疗具有高度耐药性，这促使人们迫切需要寻找新的治疗靶点来治疗高级别胶质瘤。我们之前发现Notch通路是干细胞维持和细胞命运决定的关键，是这样一个靶标。Notch-1及其配体在胶质瘤中过度表达和活跃，阻断其表达在人胶质瘤细胞中是致命的。由于Notch抑制在干细胞中的突出作用，它可能特别有希望。最近来自我们和其他人的证据表明，胶质瘤和其他癌症含有一个负责肿瘤发生的肿瘤干细胞的小治疗抵抗亚群。这些胶质瘤干细胞对标准疗法的耐药性使得找到其他方法来靶向它们变得至关重要。早期证据表明，阻断正常干细胞中必不可少的通路，如Hedgehog， Wnt和Notch，可能对肿瘤干细胞具有治疗作用。迄今为止报道的唯一Notch抑制剂是γ -分泌酶抑制剂（GSIs），它阻断Notch加工所必需的酶裂解。然而，Notch在被γ -分泌酶切割之前也会被α -分泌酶切割，这表明该通路中存在另一个脆弱点。我们的初步数据首次表明，α -分泌酶抑制剂（ASIs）可阻断Notch活性，并抑制胶质瘤干细胞的生存能力。在这项应用中，我们建议系统研究ASIs作为一类未开发的Notch抑制剂，对胶质瘤细胞，包括胶质瘤肿瘤干细胞（GTSCs）具有活性。在Aim #1中，我们比较了特异性和非特异性ASIs与gsi作为Notch抑制剂，并对它们的组合进行了评估。此外，我们确定了降解Notch的泛素连接酶FBXW7的水平是否影响胶质瘤细胞对分泌酶抑制剂抑制Notch的敏感性。为了进一步研究ASIs作为潜在的抗胶质瘤疗法，我们在aim #2中确定了ASIs对胶质瘤形成中其他主要途径的影响。在Aim #3中，我们比较了ASIs和gsi对GTSC活力和分化的影响，并评估恢复Notch活性是否可以挽救表型。我们在Aim #4中评估了局部输送ASI和GSI作为小鼠颅内GTSC模型的治疗方法，单独或联合放疗。此外，在Notch控制下表达荧光素酶的胶质瘤细胞系将用于通过局部递送Notch抑制剂来实时跟踪Notch活性。实验的成功完成将验证ASIs是一类新的Notch抑制剂，并表征其对胶质瘤和gtsc的作用。这项工作将确定这些药物作为神经胶质瘤患者的潜在治疗方法，对其他类型的癌症也有影响。公共卫生相关性：胶质瘤是最常见和最致命的脑肿瘤。它们对标准化疗和放疗具有高度耐药性，我们和其他人最近的研究表明，这可能部分是由于治疗耐药的“胶质瘤肿瘤干细胞”（GTSCs）亚群的存在。我们之前确定Notch通路是胶质瘤的一个有希望的治疗靶点。靶向Notch等干细胞通路可能是抑制肿瘤干细胞群的一种手段。在这个提议中，我们将研究α -分泌酶抑制剂作为一类新的Notch抑制剂，对胶质瘤肿瘤干细胞具有治疗活性。

DESCRIPTION (provided by applicant): Despite notable advances in treating other cancers during the last two decades, brain tumors remain a daunting therapeutic challenge. These tumors are highly resistant to standard treatment with radiation and chemotherapy, and this has driven an urgent search for novel therapeutic targets in high-grade gliomas. We previously showed the Notch pathway, key in stem cell maintenance and cell fate determination, to be one such target. Notch-1 and its ligands are over-expressed and active in gliomas, and blocking their expression is lethal in human glioma cells. Notch inhibition may be especially promising because of its prominent role in stem cells. Recent evidence from us and others indicates that gliomas and other cancers harbor a small therapy-resistant subpopulation of tumor stem cells responsible for tumorigenesis. The resistance of these glioma tumor stem cells to standard therapies makes it critical to find other means to target them. Early evidence suggests that blockade of pathways essential in normal stem cells, such as Hedgehog, Wnt, and Notch, may have therapeutic utility against tumor stem cells. The only Notch inhibitors reported to date are the gamma-secretase inhibitors (GSIs), which block an enzymatic cleavage necessary for Notch processing. However, Notch is also cleaved by an alpha-secretase prior to its cleavage by gamma-secretase, suggesting another vulnerable point in the pathway. Our preliminary data indicate for the first time that alpha-secretase inhibitors (ASIs) block Notch activity and also inhibit the viability of glioma tumor stem cells. In this application, we propose the systematic investigation of ASIs as an unexplored class of Notch inhibitors with activity against glioma cells, including glioma tumor stem cells (GTSCs). In Aim #1, we compare specific and non-specific ASIs with GSIs as Notch inhibitors and also assess them in combination. In addition, we determine if levels of the ubiquitin ligase FBXW7, which degrades Notch, affect sensitivity of glioma cells to Notch inhibition by secretase inhibitors. To further investigate ASIs as potential anti-glioma therapies, we determine in aim #2 the effects of ASIs on other pathways central in gliomagenesis. In Aim #3, we compare ASIs and GSIs for their effects on GTSC viability and differentiation and evaluate if restoration of Notch activity can rescue the phenotype. We assess local delivery of an ASI and GSI as therapies in mouse intracranial GTSC models in Aim #4, alone and in combination with radiotherapy. Additionally, a glioma line with luciferase expressed under Notch control will be used to track Notch activity real-time in vivo with local delivery of Notch inhibitors. Successful completion of the proposed experiments will validate ASIs as a novel class of Notch inhibitors and characterize their effects on gliomas and GTSCs. This work will establish these agents as potential therapies in patients with gliomas, with implications for other cancer types as well. PUBLIC HEALTH RELEVANCE: Gliomas are the most common and most lethal brain tumors. They are highly resistant to standard chemotherapy and radiation, and recent work by us and others suggests that this could in part be due to the presence of a subpopulation of therapy-resistant "glioma tumor stem cells" (GTSCs). We previously identified the Notch pathway as a promising therapeutic target in gliomas. Targeting stem cell pathways such as Notch may be a means to inhibit the tumor stem cell population. In this proposal we will investigate alpha-secretase inhibitors as a novel class of Notch inhibitors with therapeutic activity against glioma tumor stem cells.