Abstract PR011: Downregulation of the HIF2a target cyclin D1 underlies the efficacy of the HIF2a inhibitor belzutifan in kidney cancer

Most clear cell renal cell carcinomas (ccRCCs) are caused by loss of the pVHL tumor suppressor function. Inactivation of pVHL leads to increased accumulation of HIF2a, which drives ccRCC in preclinical models. HIF2a interacts with ARNT and functions as a heterodimeric transcription factor. HIF2a can activate hundreds of downstream target genes, including many that are suspected of playing a role in ccRCC pathogenesis based on their biological functions. Peloton Therapeutics developed a specific allosteric HIF2a inhibitor named PT2399, which disrupts the HIF2a-ARNT interaction and silences the HIF2a transcription program. Furthermore, PT2399 suppresses the growth of ccRCC cells in in-vitro and in-vivo assays. A more advanced version of PT2399, Belzutifan, was recently approved for treating tumors in patients with germline VHL mutations (VHL disease) and is in Phase 3 testing for sporadic ccRCCs. Since HIF2a is a transcriptional activator, we hypothesized that PT2399’s ability to suppress ccRCC is linked to repression of one or more HIF2a target genes. To identify these genes, we performed a pooled whole genome CRISPR activation (CRISPRa) screen in the presence or absence of PT2399. This screen identified multiple genes (“hits”) that, when activated, likely conferred resistance to PT2399. To further differentiate between direct vs indirect HIF2a targets that confer resistance to PT2399, we performed nascent RNA-seq (Pro-seq) after acute treatment of ccRCC cells with PT2399. Comparison of the CRISPRa and Pro-Seq data, together with secondary validation assays, identified CCND1, which encodes Cyclin D1, as a direct target gene of HIF2a that, when activated, confers complete resistance to PT2399 in multiple ccRCC cell lines. Additionally, we showed that wild-type Cyclin D1 (WT), but not a mutant (KE) that fails to activate CDK4/6, confers resistance to PT2399. Interestingly, Cyclin D1 overrides the growth inhibitory effect of PT2399 in ccRCC cells irrespective of pRB status. To identify the relevant Rb-independent substrates of Cyclin D1, we performed IP-MS experiments in RB1-/- ccRCC cells under substrate trapping conditions. Our preliminary data from these experiments suggests that RBL1 (p107) and RBL2 (p130) substitute for pRB in RB1-/- ccRCC cells. Our findings provide further support for CDK4/6 as a therapeutic target in ccRCC. Citation Format: Nitin H. Shirole, Devishi Kesar, William R. Sellers, William G. Kaelin Jr. Downregulation of the HIF2a target cyc