There are still unmet clinic needs for more effective and long-lasting targeted therapy against BRAF in advanced melanoma skin cancer and other BRAF-mutated cancers. This exploratory project aims to further understand molecular regulation of BRAF and to develop such new therapy drugs that are mechanistically different than the current clinic drugs. The long-term goal is to develop luteolin analogues as a new class of therapeutic agents on MAPK-dependent cancers. The short-term goal of this project is to understand how luteolin induces BRAF degradation. Luteolin inhibited melanoma cell growth in vitro and in vivo, and exhibited substantial synergistic effect with Vemurafenib in all melanoma cell lines examined. Luteolin inhibited BRAF kinase activity in a cell-free system, and induced BRAF degradation in melanoma cells, which led to down- regulation of the MAPK pathway. Luteolin was docked to Cys532 in BRAF crystal structure by a computational docking analysis. It is thus hypothesized that luteolin inhibits melanoma tumor growth primarily via direct binding to BRAF protein at the druggable Cys532 residue, resulting in proteasome-mediated BRAF degradation. BRAF-wt and BRAF-mt may use different mechanisms. Two specific aims are proposed: Specific Aim 1: To identify the role of Cys532 in BRAF stability and kinase activity. The role of Cys532 in BRAF function is largely unknown but was computationally predicted as “druggable”. In this aim Cys532 will be genome-edited to become a glycine (C532G) or a Tyr (C532Y) in BRAFwt/wt and BRAFV600E/V600E backgrounds. BRAF protein stability and kinase activity will be analyzed by western blots in the various edited genetic backgrounds. Whether luteolin-induced ROS play a role in BRAF stability and activity through oxidation of Cys532. Additionally, drug sensitivity (luteolin, Vemurafenib or combination of luteolin/Vemurafenib) will be assayed and compared in these genetic backgrounds in vitro and in vivo. Drug Affinity Responsive Target Stability (DARTS) assay will be used to evaluate direct binding of luteolin to BRAF with various mutations. Specific Aim 2: To identify the mechanism of luteolin-induced BRAF degradation. Luteolin-induced BRAF degradation is proteasome-dependent but a specific E3 ligase is not identified. Anaphase-Promoting Complex (APC) and the SKP1/CUL1/F-Box complexes (as well as USP28 which counteracts SKP activity) were reported to be involved in BRAF protein degradation. In this aim small molecule inhibitors specific for APC or for SCF will be used to determine luteolin-mediated E3 ligase for BRAF destruction. Lentiviral shRNA approach will be used at last to validate the specific E3 ligase complex by knocking down the core component of the ligases (APC10 or SKP1). In parallel, we identified USP35 and six additional E3 ligases through RNA-Seq as potential luteolin targeted enzymes which will be examined for their impact on BRAF degradation. Completion of these aims will enable us to initiate...