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.