PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is a deadly primary brain malignancy with limited therapeutic options. Tumor progression is thought to be driven by stem cell-like cells that evade conventional chemoradiotherapy and anti- angiogenic treatment. Indeed, anti-angiogenic therapy and subsequent worsening of tumor oxygenation promote hypoxia-resistant stem cell phenotypes that lead to further tumor progression. However, our understanding of mechanisms that underlie both GBM stem cell (GSC) behavior and its regulation by oxygen tension remains incomplete. In our effort to identify novel targetable mediators of the GSC phenotype, we recently discovered that GPR133 (ADGRD1), an orphan member of the adhesion family of G protein-coupled receptors, is necessary for initiating tumor growth in vitro and in vivo, both GSC properties, in part by triggering signaling mechanisms that increase cytoplasmic cAMP and lead to transcription of genes necessary for “stemness”. While GPR133 is absent from normal brain tissue, it is expressed with full penetrance in all GBM specimens tested, regardless of molecular subtype. On the basis of these findings, we hypothesize that GPR133 is a critical component of tumor growth by supporting the GSC phenotype. We, therefore, believe that GPR133 inhibition represents a novel and appealing therapeutic strategy in GBM that merits further testing and development. We now seek to expand on our published findings and use patient-derived GBM models to elucidate basic mechanisms of action of GPR133. Aim 1 will test the hypotheses that GPR133 identifies GBM stem cells and its knockdown in tumor xenografts slows tumor growth and prolongs survival. Aim 2 will build on our finding that, within each tumor, GPR133 expression is highest in the most hypoxic regions, suggesting regulation by oxygen tension. More specifically, we will determine the effect of intratumoral fluctuations in oxygenation on GPR133 expression by correlating mRNA and protein levels with tumor vascularity and oxygenation using targeted intraoperative biopsies of patient tumors. In addition, Aim 2 will determine whether GPR133 knockdown synergizes with cediranib, an anti-angiogenic agent, to prevent tumor progression after aggravation of tumor hypoxia. Finally, Aim 3 will determine the relative contribution of canonical G protein signaling initiated by GPR133 and transduced by cAMP and its effectors RAP and PKA, and adhesion mediated by GPR133’s long N- terminal ectodomain, to the transcriptional regulation of genes that support the GSC phenotype. The proposed studies will mechanistically clarify GPR133’s role in tumor progression, including in hypoxia exacerbated by anti-angiogenic therapy. The r...