Requirement for Cyclin D1 Underlies Cell Autonomous HIF2-Dependence in Kidney Cancer

Abstract Inactivation of the VHL gene stabilizes HIF2a, which drives clear cell renal carcinoma (ccRCC). The HIF2a inhibitor belzutifan is approved for ccRCC treatment, but de novo and acquired resistance are common. HIF2a, bound to ARNT, transcriptionally activates many genes. We performed CRISPRa screens in HIF2a-dependent ccRCC lines treated with a belzutifan analog to identify HIF2a-responsive genes that confer cell-autonomous belzutifan resistance when not downregulated. Sustaining the expression of the HIF2a target gene CCND1, encoding Cyclin D1, promoted HIF2a-independence/belzutifan resistance. This activity requires Cdk4/6 activation by Cyclin D1, but is not solely due to phosphorylation of the canonical Cyclin D1 target, pRB. Indeed, ccRCC lines lacking all three pRB family members remained at least partially HIF2a-dependent. In this context, however, a kinase-defective Cyclin D1 variant partially overrode belzutifan’s antiproliferative effects, suggesting that ccRCC promotion by Cyclin D1 requires the phosphorylation of pRB paralogs and one or more kinase-independent Cyclin D1 activities.