PROJECT SUMMARY Anti-angiogenic therapy holds much promise for the treatment of malignancies like glioblastoma (GBM), a devastating brain cancer for which effective treatments are badly needed. Based on encouraging clinical trial results, in 2009, the anti-angiogenic VEGF-neutralizing antibody bevacizumab was granted accelerated FDA approval for recurrent GBM treatment. However, while the initial responses to anti-angiogenic therapy are often significant, subsequent randomized trials have shown that these agents have limited durations of response. Many tumors, after responding initially, develop acquired invasive resistance, a rapidly progressive state with a poor prognosis. Mouse models suggest that resistance to anti-angiogenic therapy likely reflects post- transcriptional protein modifications that are more readily generated than the mutations that cause traditional chemotherapy resistance. Along these lines, during the past four years of funding, we have shown that bevacizumab-induced VEGF depletion causes GBM cells to release receptor tyrosine kinase c-Met and β1 integrin from VEGFR2 sequestration, enabling these two receptors to form a powerful structural complex in which c-Met displaces α5 integrin from its β1 binding site due to greater affinity and the c-Met/β1 complex exhibits increased affinity than α5β1 integrin for fibronectin. To advance these findings, the goal of this grant renewal is to investigate the hypothesis that invasive resistance to anti-angiogenic therapy can be overcome by targeting the interaction between c-Met and β1 integrin. We will investigate this hypothesis within the following Specific Aims: Aim 1 - Investigate mechanisms by which VEGF depletion drives c-Met/β1 complex- mediated invasiveness in bevacizumab-resistant GBM; Aim 2 – Determine if the c-Met/β1 complex gives rise to specific cytoskeletal changes that drive invasive bevacizumab resistance in GBM; and Aim 3 - Identify therapies that inhibit the binding of c-Met and β1 integrin in bevacizumab-resistant GBM. We will carry out these studies using unique tools and innovations developed in my lab, including our novel in vivo models of anti-angiogenic therapy resistance, along with 3D bioengineered systems for studies of tumor cell invasion and small molecule inhibitor libraries created by our collaborators. These tools will be analyzed using the latest techniques, including CRISPR gene editing and mass spectrometry-based immuno-precipitation proteomics to assess the impact of c-Met-β1 binding. Successful completion of this project would define central mechanisms of resistance to anti-angiogenic therapy driven by prolonged VEGF depletion reversing the normal invasion suppressing effects of VEGF and would identify agents targeting invasive resistance to anti-angiogenic therapy. Therefore, we expect these studies to offer insight into the double-edged sword of anti-angiogenic therapy by revealing adverse effects of prolonged VEGF blockade, and could ultimately a...