The long term objective of this proposal is to improve the therapy of glioblastoma multiforme (GBM) by defining pathways of resistance to the death ligand TRAIL. TRAIL is a pro-apoptotic peptide that binds TRAIL receptors and induces a tumor-selective cell death. Although many GBM are susceptible to TRAIL, resistance is common, and contributes to the failure of therapies that rely on TRAIL-based killing. TRAIL resistance in GBM cells is associated with PTEN loss, Akt activation, and increased production of the anti-apoptotic protein FLIPs. PTEN loss and Akt activation, however, also contribute to TRAIL resistance by decreasing the ubiquitination and destruction of the FLIPs protein. The means by which PTEN regulates the ubiquitination of FLIPs have not been described, although we have identified two involved proteins; an E3 ubiquitin ligase called AIP4 that directly interacts with, ubiquitinates, and destabilizes FLIPs, and a ubiquitin-removing enzyme called USP8 whose degradation in response to PTEN loss leads to FLIPs stabilization. We do not currently understand if or how these proteins may be linked to each other and to PTEN, nor do we understand the relevance of these proteins for resistance to TRAIL-based therapies. We do know, however, that USP8, AIP4, and FLIPs all undergo co-ordinated changes in ubiquitination following PTEN loss, and that these changes in ubiquitination have the potential to alter protein function (if ubiquitin is added as a single peptide or as chains linked through lysine 63 of ubiquitin) or stability (if added as chains linked through the lysine 48 position of ubiquitin). Based on our preliminary data, we believe that PTEN inactivation sets off a cascade of ubiquitin reactions that ultimately stabilizes FLIPs and confers TRAIL resistance. Specifically we hypothesize that PTEN loss induces an Akt-mediated phosphorylation, K48 polyubiquitination, and degradation of USP8. In the absence of the deubiquitinating activity of USP8, the USP8 target and E3 ligase AIP4 is left in an inactive (mono- or K63-polyubiquitinated) state that is incapable of ubiquitinating/destroying its target FLIPS and that leads to TRAIL resistance in GBM. To test this hypothesis we will 1: Determine if direct Akt-mediated phosphorylation of USP8 increases USP8 ubiquitination, USP8 degradation, and TRAIL resistance. 2: Identify the Akt-dependent E3 ligase responsible for USP8 ubiquitination. 3: determine if Akt activation causes distinct ubiquitination patterns that in turn bring about USP8 degradation, AIP4 inactivation, FLIPS stabilization, and TRAIL resistance, and 4: Determine if USP8 alters AIP4 interactions and/or activity, and if this is critical for USP8-mediated FLIPs destabilization and reduced TRAIL resistance. Because TRAIL is a promising therapeutic agent on its own, as well as an important component of how the immune system and immune-based therapies eliminate GBM cells, the present work will provide insight as to how to stratify patients for TRAIL- or immune-based therapies, how to circumvent the TRAIL resistance commonly seen in GBM, and in general how to create more effective TRAIL-based therapies. The present work will also serve as a template for investigating how PTEN controls global protein stability, and will set the stage for the development of agents that, by selectively targeting various DUB/E3 pairs, can selectively control target protein function and PTEN tumor suppressor action.