1. Identification of novel HIF-2 inhibitors. Prior studies have shown that normoxic stabilization of HIF1-alpha alone, while capable of mimicking some aspects of VHL loss, are not sufficient to reproduce tumorigenesis, indicating that it may not be the most critical oncogenic substrate of VHL protein. To isolate compounds that selectively modulate HIF2-alpha for use as research tools and drug development leads, a cell-based high throughput screening assay of the NCI Natural Products Repository was developed in collaboration with Dr. Tawnya McKee in the Molecular Targets Laboratory (NCI/CCR/MTL). A series of reporter plasmids were designed that placed tandem copied of the minimal hypoxia response element of the VEGF gene in control of the gene encoding firefly luciferase. These plasmids were transfected into the VHL-and HIF1-negative RCC tumor-derived cell line 786-0 and derived cell lines were optimized for use in high-throughput detection systems in the MTL. Counter screens for global transcriptional repression as well as cell toxicity were components of the final screening strategy. Leads from screening of the NCI Natural Products Repository were chromatographically separated into component structures yielding approximately 40 pure compounds with micromolar or submicromolar IC50 values, &gt;80% inhibition and &lt;5% cell toxicity. These hits were then re-screened for VEGF protein secretion by 786-0 in the UOB. A majority of the compounds identified in the initial screen showed also modulation of VEGF secretion; dose response curves of the 6 most active compounds revealed 2 compounds with low micromolar inhibition of VEGF protein secretion. These leads, as well as the 30 remaining most active hits have been tested in PCR-based assays for their affects on a panel of 7 HIF-regulated genes in three clear cell RCC-derived cell lines: 786-0 (HIF2 expression only), RCC4 (equal expression of HIFs 1 and 2) and SK-RC-28 (engineered for suppression of HIF2). This evaluation has distinguished compounds with activity toward HIF1, 2 or both. Recent biological characterization of these compounds has provided biochemical confirmation of activity against the HIF2 promoter, as well as preliminary bioassays for proliferation, survival, motility and anchorage independent growth. 2. Further preclinical assessment of inhibitors of the VHL/HIF/mTor axis identified in Aim 1 in animal models will depend on the availability of adequate quatitites of the compounds, and will assess toxicity, PK and efficacy in tumor xenograft and UOB-developed mouse models of RCC. Current work toward this goal utilizes commercially available agents targeting components of the VHL/HIF signaling axis and angiogenic pathways, as well as the experimental drug candidates NK1/3S and VEGF/3S developed in our group that are active against the hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) pathways. These studies will interrogate the integration of growth factor pathways with those driving metabolic pathways toward anaerobic glycolysis (the Warburg effect) in clear cell carcinoma as well as RCC tumors associated with hereditary papillary RCC, BHD and HLRCC. Our research strategy exploits genetically altered and engineered cultured cell models derived from UOB patient tumors, as well as transgenic and knock-out mouse models of these diseases also developed in UOB.