Our laboratories have focused on understanding how the EGF receptor (EGFR) and its signaling partner, the GTP-binding/protein crosslinking enzyme tranglutaminase-2 (TGase-2), contribute to aggressive cancers, and discovered that TGase-2 enhances EGFR signaling by blocking its degradation by c-Cbl. The goals of CA201402 were then to examine whether TGase-2 provides a similar protective effect for the EGFR oncogenic variant, EGFRvIII, in glioma stem cells (GSCs), and if these proteins are important for the actions of their extracellular vesicles (EVs). Our efforts have resulted in a number of new discoveries, which form the basis of this renewal application. These include the finding that TGase-2 function is coupled to its ability to adopt two distinct conformational states, a GTP-bound closed state that protects EGFRs from Cbl-catalyzed degradation, and a protein crosslinking-competent open-state that binds to large EVs, called microvesicles (MVs), and is necessary for their ability to influence the tumor microenvironment and stimulate angiogenesis. However, when TGase-2 is induced to adopt an open state within cancer cells, it causes cell death. Although we found that EGFRvIII does not require TGase-2 to protect it from Cbl-catalyzed ubiquitylation, we discovered EGFRvIII expression is coupled to glutamine metabolism which is elevated in aggressive cancer cells. Moreover, we found aggressive cancer cells generate large numbers of small EVs (exosomes) that contain the immune checkpoint ligand, PD-L1, and the cell survival protein Survivin, due to their elevated glutamine metabolism and their down-regulation of Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase. We will now set out to understand the consequences of these discoveries for the growth and survival of GSCs, through the efforts of two laboratories with complimentary expertise in cancer cell signaling (Cerione) and translational studies of GSCs (Nakano), as follows: 1) Determine how to exploit the openstate conformation of TGase-2 to inhibit cancer cell viability. We will establish how open-state TGase-2 causes cancer cells to undergo cell death and develop methods to stabilize that state as a potential therapeutic strategy. 2) Understand how EGFRvIII expression in aggressive GSCs is coupled to glutamine metabolism. We will determine why inhibiting glutamine metabolism markedly affects the cellular levels of EGFRvIII expression, and whether EGFRvIII expression and signaling stimulate the production of MVs containing this oncogenic variant and TGase-2. 3) Understand the connection between the expression of EGFRvIII and TGase-2 in aggressive GSCs and the shedding of large numbers of exosomes with unique protein cargo. We will establish whether down-regulation of SIRT1 in GSCs, and signaling pathways involving EGFRvIII and/or TGase-2, are responsible for their ability to produce large numbers of exosomes enriched in PD-L1 and Survivin. These studies will provide new insights into the development of aggressive cancers such as high-grade gliomas as well as highlight novel therapeutic targets for aggressive disease.