58Dissecting the interplay between Bap1 and Pbrm1 in renal cell carcinoma

Abstract Background Polybromo 1 (PBRM1) and BRCA1-associated protein 1 (BAP1) are tumor suppressor genes frequently mutated in clear cell renal cell carcinoma (ccRCC) with distinct contributions to tumor grade and aggressiveness that are mutually exclusive. Interestingly, rare double-mutant tumors are particularly aggressive and associated with worse prognosis. Methods To understand the effects of simultaneous inactivation of PBRM1 and BAP1 we generated conditional double mutant mouse models. We introduced variant LoxP sites flanking essential Bap1 sequences in Pbrm1F/F zygotes using CRISPR/Cas9 overcoming thereby barriers associated with genetic linkage. We then investigated the developmental and oncogenic consequences of Bap1/Pbrm1 co-inactivation using different Cre drivers. Next, we evaluated the impact of simultaneous inactivation of Bap1 and Pbrm1 in the kidney using a Pax8-Cre driver (double knockout [DKO mice]). To further explore the role of Bap1 and Pbrm1 in ccRCC development, we generated mice with simultaneous inactivation of the von Hippel-Lindau (Vhl) gene (triple knockout [TKO mice]). To probe ccRCC pathogenesis, we performed both RNA-seq and GeoMx spatial transcriptomic profiling in DKO and TKO mice. Results In the embryonic model, simultaneous inactivation of Bap1 and Pbrm1 in embryos using a CAG-Cre accelerated embryonic lethality. Developmental defects were observed as early as E7.5, compared to the previous established developmental defects for Bap1 and Pbrm1 loss at E8.5 and E11.5, respectively. This finding supports the idea that simultaneous inactivation of Bap1 and Pbrm1 accentuates developmental phenotypes. Using Pax8-Cre driver, compared to Bap1F/F and Pbrm1F/F mice, we found that kidneys of DKO mice developed cystic tumors, suggesting that loss of Bap1 and Pbrm1 alone is sufficient for tumorigenesis. Interestingly, TKO mice developed aggressive ccRCC tumors that differed from those observed in DKO mice. Interestingly, we observed that tumor grade was a more important driver of gene expression than genotype. In addition, while Pbrm1-deficient tumors exhibited a metabolic shift from oxidative phosphorylation to glycolysis (consistent with the Warburg effect), Bap1 loss was associated with immune cell activation and mesenchymal features. GeoMx spatial transcriptomic analyses showed that DKO tumors were enriched for T-effector and complement cascade signatures, while TKO tumors showed increased angiogenesis and fatty acid metabolism. Conclusions Together, our results suggest that BAP1 and PBRM1 are infrequently mutated together because of developmental synthetic lethality, but when co-occurring with VHL loss, can drive aggressive tumor phenotypes. This study highlights the genetic crosstalk between key tumor suppressors in shaping RCC evolution and heterogeneity.