Abstract 1719: VHLloss enables immune checkpoint blockade therapy by boosting interferon response

Abstract Background: The von-Hippel Lindau (VHL) as a part of E3 ubiquitin ligase is a key regulator of hypoxia induced factors (HIFs). It is also characterized as a tumor suppressor gene mutated in up to 80% clear cell renal cell carcinoma (ccRCC) cases. Immune checkpoint blockade (ICB) therapy has shown improved ccRCC treatment outcomes. However, the biological roles of the VHL gene in ICB therapy are to be explored. Methods: The association between VHL gene mutation and overall survival of patients receiving ICB treatments was determined by integrating multiple human datasets. CRISPR/Cas9 was used to generate VHL knockout (KO) for in vivo tumor growth and in vitro mechanistic studies. The anti-tumor responses of genetic VHL loss in combination with anti-PD1 treatment were evaluated in three syngeneic murine tumor models. Tumor infiltrated lymphocytes and intratumoral T-cell receptor (TCR) diversity were analyzed by flow cytometry and TCR sequencing, respectively. Mitochondrial defects resulted from VHL loss were tested by JC-1 mitochondrial membrane potential assay, immunofluorescence microscopy, and qRT-PCR. VHL loss-mediated downstream signaling were further examined by western blot and bulk RNA sequencing. Results: The substantial overall survival benefits among ICB-treated patients withVHL mutations across different cancers, including RCC, in multiple human cohorts. Genetic knockout of VHL enhanced anti-PD1 therapy in three murine tumor models, including those of non-RCC origins. Moreover, VHL loss caused significant infiltration of immune effector cells and increased diversity of intratumoral TCR repertoire. VHL deficiency resulted in HIFα accumulation, increased expression of pro-mitophagy factor BNIP3, and impaired mitochondrial membrane potential that led to the cytoplasmic leakage of mitochondrial DNA (mtDNA), triggering cGAS-STING activation and type I interferon production. HIFα overexpression showed similar results as demonstrated in VHL-KO cells. In contrast, double knockdown of VHL and HIFα reversed VHL-loss induced BNIP3 upregulation, rescued mtDNA leakage to the cytoplasm, and disrupted constitutive activation of cGAS-STING signaling. Furthermore, blocking type I interferon signaling abrogated VHL-deficiency induced tumor suppression. Conclusion: Our study uncovered a novel role for VHL loss in creating an immunogenic tumor microenvironment through the activation of mtDNA-cGAS-STING pathway and type I interferon signaling in tumor cells via VHL-HIFα-BNIP3 axis, making them susceptible to ICB therapy. This finding provides a mechanistic explanation for ccRCC responsiveness to ICB therapy despite a moderate tumor mutation burden and suggests potential therapeutic strategies targeting ccRCC and other VHL-deficient tumors based on constitutive cGAS-STING and type I interferon activation. Citation Format: Meng Jiao, Xuhui Bao, Mengjie Hu, Dong Pan, Jonathan Kim, Xinjian Liu, Fang Li, Chuan-Yuan Li. VHLloss enables immune checkpoint blockade therapy by boosting interferon response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1719.