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

描述(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步，但脑瘤仍然是一个令人望而生畏的治疗挑战。这些肿瘤对放射和化疗的标准治疗具有高度抵抗力，这促使人们迫切地寻找高级别胶质瘤的新治疗靶点。我们之前展示了Notch通路，干细胞维持和细胞命运决定的关键，就是这样的靶子之一。Noch-1及其配体在胶质瘤中过度表达和活跃，阻断其表达对人脑胶质瘤细胞具有致命性。缺口抑制可能特别有希望，因为它在干细胞中具有突出的作用。来自我们和其他人的最新证据表明，胶质瘤和其他癌症含有一小部分负责肿瘤发生的肿瘤干细胞亚群，这些细胞对治疗具有抵抗力。这些胶质瘤干细胞对标准疗法的抵抗力使得寻找其他方法来靶向它们至关重要。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。到目前为止，唯一报道的Notch抑制剂是伽马分泌酶抑制剂(GSI)，它阻止了Notch加工所必需的酶切割。然而，Notch在被伽马-分泌酶切割之前也被α-分泌酶切割，这表明这是该途径中的另一个脆弱点。我们的初步数据首次表明，α-分泌酶抑制剂(ASIS)可以阻断Notch活性，也可以抑制胶质瘤干细胞的活性。在这一应用中，我们建议系统地研究ASIS作为一类未知的Notch抑制剂，对包括胶质瘤干细胞(GTSCs)在内的胶质瘤细胞具有活性。在目标1中，我们将特异性和非特异性ASIS与GSI作为Notch抑制剂进行比较，并对它们联合进行评估。此外，我们还确定了降解Notch的泛素连接酶FBXW7的水平是否会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治疗方法，我们在AIM#2中确定了ASIS对胶质瘤形成的其他中心途径的影响。在目标3中，我们比较ASIS和GSI对GTSC活性和分化的影响，并评估Notch活性的恢复是否可以挽救GTSC的表型。在AIM#4中，我们评估了ASI和GSI作为治疗方法在小鼠颅内GTSC模型中的局部应用，包括单独和联合放射治疗。此外，一种在Notch控制下表达荧光素酶的胶质瘤株将被用于通过局部输送Notch抑制剂来实时跟踪体内的Notch活性。拟议实验的成功完成将验证ASIS作为一种新型的Notch抑制剂，并表征它们对胶质瘤和GTSCs的作用。这项工作将确立这些药物作为胶质瘤患者的潜在治疗方法，并对其他类型的癌症也有影响。与公共卫生相关：胶质瘤是最常见和最致命的脑肿瘤。它们对标准的化疗和放射具有高度的抵抗力，我们和其他人最近的工作表明，这可能部分是由于存在一个亚群的治疗耐药的“胶质瘤干细胞”(GTSCs)。我们先前发现Notch通路是治疗胶质瘤的一个很有前途的靶点。靶向干细胞途径，如Notch，可能是抑制肿瘤干细胞数量的一种手段。在这项提案中，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.