Project Summary HIF2α is arguably the most important driver of clear cell renal cell carcinoma (ccRCC), the most common kidney cancer type. Discovered at UT Southwestern Medical Center (UTSW) and regarded as undruggable, structural studies revealed a vulnerability that was exploited leading to the identification of small molecule inhibitors and the founding of a biotech company, Peloton Therapeutics, Inc., which developed several analogues (PT2385, PT2399 and PT2977). PT drugs bind to HIF2α leading to its dissociation from its obligatory partner HIF1β, thereby inhibiting HIF2-mediated transcription. Preclinical and clinical work during the previous funding period advanced the field significantly. Using tumorgraft (TG) models of patient ccRCCs transplanted in mice, we showed that: HIF2 is a valid target; approximately 50% of ccRCC are dependent on HIF2; HIF2-sensitive tumors are characterized by high HIF2α, which may serve as a biomarker; and resistance develops as a result of acquired HIF2α mutations. In addition, we led accrual to the phase 1 trial, which showed that PT2385 was well tolerated and active. Furthermore, we showed that: (i) PT2385 specifically targeted HIF2 in patient tumors (but not HIF1); (ii) HIF2 targeting resulted in inhibition of HIF2 target genes; and (iii) prolonged therapy led to the acquisition of resistance mutations we predicted in TGs. These data identified HIF2α as the first core dependency in ccRCC. The success of the program culminated with the approval by the FDA of PT2977 (belzutifan). For the next funding period, we seek to co-develop: (i) an siRNA-based HIF2α drug (siHIF2α) that targets both wild-type and resistant-mutant HIF2α; and (ii) a molecular imaging tool that can enable non-invasive HIF2α monitoring in patients. Developed by Arrowhead Pharmaceuticals Inc., siHIF2α is a synthetic, stabilized, chemically modified, double-stranded RNAi trigger specifically targeting HIF2α mRNA conjugated to a high-affinity ligand for integrins αvβ3 and αvβ5, which are broadly expressed in ccRCC. siHIF2α will be co-developed with a novel imaging tool we generated to enable HIF2α quantification, and which will be deployed to measure siRNA-mediated target depletion. We leveraged the high specificity of PT2385 and by substituting a native fluorine atom converted it into a PET radiotracer ([18F]PT2385). Since the original submission a year ago, we received an IND for ([18F]PT2385), obtained IRB approval, and began dosimetry studies in humans. We also advanced the siHIF2α phase 1 trial to the last planned cohort revealing good tolerability, showing that siHIF2α downregulates HIF2α in patient tumor biopsies, and providing preliminary evidence of activity. Having successfully launched a HIF2α inhibitor, now FDA approved, we propose to develop a second-generation inhibitor targeting wild-type and drug- resistant HIF2α. If successful, this may lead to a new drug for ccRCC and possibly the first siRNA-based therapeutic in oncology.