PROJECT SUMMARY The Wnt pathway is frequently dysregulated in many cancers, underscoring it as a therapeutic target. Although Wnt inhibitors appear promising in many preclinical studies, they have failed uniformly in clinical trials. Molecular mechanisms of resistance are poorly defined. Further dissection of the precise mechanisms of WNT pathway activation in specific tumor types is needed to develop new WNT pathway inhibitors with less toxicity. The axonal guidance program Sema3C/PlxnD1 promotes the self-renewal and tumorigenicity of glioma stem-like cells (GSCs), but the underlying mechanisms are unclear. Our data now suggest that Sema3C/PlxnD1 signaling functions as an alternative activator of canonical Wnt signaling. Importantly, Sema3C-driven Wnt signaling occurred despite suppression of Wnt ligand secretion, suggesting that Sema3C may drive canonical Wnt signaling independent of Wnt binding to its receptors. As Sema3C/PlxnD1 signaling is used in over 85% of GBM, it may represent an important mechanism of resistance to upstream Wnt pathway inhibitors. Our data support that Sema3C/PlxnD1 signaling regulates two critical aspects Wnt signaling: beta-catenin stability and nuclear translocation. In this proposal, we aim to identify molecular mechanisms by which Sema3C/PlxnD1 regulate canonical Wnt signaling. Additionally, we aim to assess the therapeutic impact of targeting Sema3C signaling to improve sensitivity to upstream Wnt inhibitors in mouse models of GBM. These studies will provide a novel therapeutic strategy to achieve clinically significant Wnt pathway inhibition in GSCs potentially without the toxicity of currently available WNT inhibitors. These studies may be applied to other cancers including breast and prostate cancers that utilize both Sema3C and Wnt signaling.