(1101-B) Development of Cell-based Assays for the Identification and Characterization of Inhibitors of Mutant G323E Hypoxia Inducible Factor-2α (HIF-2α).
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: The role of Hypoxia Inducible Factor-2α (HIF-2α) in the hypoxia response pathway has garnered attention as a target for the treatment of von Hippel-Lindau (VHL) linked clear cell renal cell carcinoma (ccRCC). Belzutifan is a HIF-2α small molecule inhibitor, approved by the FDA in 2021, for the treatment of VHL disease. Reported objective responses to HIF-2α inhibitors tend to be long and potential mechanisms of adaptive resistance have not been reported; however, during the development of first-generation HIF-2α inhibitor, PT2385, drug resistance was observed in one patient that led to the identification of the G323E HIF-2α gatekeeper mutant. We developed cell-based assays to determine the feasibility of generating compounds that inhibit HIF-2α with the G323E mutation. Using previously reported normoxia stable HIF-2α mutations, HEK 293 cells were engineered to express stable wild type or G323E mutant HIF-2α. These cells were transfected with a hypoxia response element (HRE) luciferase reporter to measure the ability of compounds to inhibit the HIF-2α luciferase response. Identified inhibitors were further characterized by VEGF secretion in wild type or CRISPR generated 786-O G323E mutant renal cells and in a protein thermal shift assay (TSA). The assays developed provide a platform for screening against and identifying small molecule inhibitors of the G323E HIF-2α mutant.