Renal Cell Carcinoma Programmed Death-ligand 1, a New Direct Target of Hypoxia-inducible Factor-2 Alpha, is Regulated by von Hippel–Lindau Gene Mutation Status

Background Clear cell renal cell carcinomas (ccRCC) frequently display a loss of function of the von Hippel–Lindau (VHL) gene. Objective To elucidate the putative relationship between VHL mutation status and immune checkpoint ligand programmed death-ligand 1 (PD-L1) expression. Design, setting, and participants A series of 32 renal tumors composed of 11 VHL tumor-associated and 21 sporadic RCCs were used to evaluate PD-L1 expression levels after sequencing of the three exons and exon–intron junctions of the VHL gene. The 786-O, A498, and RCC4 cell lines were used to investigate the mechanisms of PD-L1 regulation. Outcome measurements and statistical analysis Fisher's exact test was used for VHL mutation and Kruskal–Wallis test for PD-L1 expression. If no covariate accounted for the association of VHL and PD-L1, then a Kruskal–Wallis test was used; otherwise Cochran–Mantel–Haenzsel test was used. We also used the Fligner–Policello test to compare two medians when the distributions had different dispersions. Results and limitations We demonstrated that tumors from ccRCC patients with VHL biallelic inactivation (ie, loss of function) display a significant increase in PD-L1 expression compared with ccRCC tumors carrying one VHL wild-type allele. Using the inducible VHL 786-O-derived cell lines with varying hypoxia-inducible factor-2 alpha (HIF-2α) stabilization levels, we showed that PD-L1 expression levels positively correlate with VHL mutation and HIF-2α expression. Targeting HIF-2α decreased PD-L1, while HIF-2α overexpression increased PD-L1 mRNA and protein levels in ccRCC cells. Interestingly, chromatin immunoprecipitation and luciferase assays revealed a direct binding of HIF-2α to a transcriptionally active hypoxia-response element in the human PD-L1 proximal promoter in 786-O cells. Conclusions Our work provides the first evidence that VHL mutations positively correlate with PD-L1 expression in ccRCC and may influence the response to ccRCC anti-PD-L1/PD-1 immunotherapy. Patient summary We investigated the relationship between von Hippel–Lindau mutations and programmed death-ligand 1 expression. We demonstrated that von Hippel–Lindau mutation status significantly correlated with programmed death-ligand 1 expression in clear cell renal cell carcinomas.