Abstract 862: Negative cellular outcomes following acute in vivo Vhl inactivation in mice

Abstract Clear cell renal cell carcinoma (ccRCC) is driven by the biallelic inactivation of the von Hippel Lindau tumor suppressor (VHL) gene, which results directly in the normoxic stabilization of hypoxia inducible factors (HIF-1 and -2). While VHL is expressed ubiquitously, its inactivation is tumorigenic only in specific tissues. We hypothesize that this tissue-specificity arises from a negative selective pressure on VHL KO cells that is tolerated in certain contexts. To assess this phenomenon, we induced Vhl deletion in vivo simultaneously across the mouse body and observed cell fate with single-cell resolution. Here we show that inducing widespread inter-organ Vhl deletion results in a massive removal of affected cells from tissue. Our study required technology that allowed us to visualize and isolate Vhl-recombined cells from tissue. For this, we developed a novel lineage marking model in which Cre-mediated excision of Vhl is coupled to knock-in of a tdTomato reporter cassette under the endogenous Vhl promoter. We used this model in conjunction with the ubiquitously-expressed UBC-CreERT2 to induce marked biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl. One week after inactivation was induced by administration of tamoxifen, there were equal numbers of tdTomato-positive cells in both genotypes, implying that initial recombination was similar and that Vhl KO did not affect immediate cell survival. Surprisingly, there was a massive reduction in the number of tdTomato-positive cells in multiple KO/KO organs after four weeks. This loss was accompanied by proliferation of tdTomato-negative cells, as assayed by BrdU incorporation, suggesting that tissues can at least partially compensate for loss of Vhl-inactivated cells. Finally, we found that the removal of cells was preceded by signs of cellular damage and stress, such as accumulation of p27, cytoplasmic translocation of HMGB1, and TUNEL-positivity in the kidney and liver. Taken together, we conclude that Vhl inactivation is inherently deleterious to cells and entrains a negative cell selection process in multiple organs. One corollary is that there must exist some conditions under which such selection might not occur; secondary mutations found in ccRCC, such as in Pbrm1, or loss of individual HIF isoforms, might provide such conditions to Vhl KO cells. Currently we are investigating the role of these additional perturbations in modifying the cellular response to Vhl loss. Our study will provide new insights into the genesis of ccRCC by redefining the mechanisms underlying the creation of pro-tumorigenic niches following Vhl inactivation. Citation Format: Samvid Kurlekar, Joanna D C Lima, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. Negative cellular outcomes following acute in vivo Vhl inactivation in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 862.