Abstract 939: A novel lineage-tracing model of Vhldeletion reveals time-dependent cellular changes in the kidney
Authors: Lima Joanna D C , Kurlekar Samvid , Pugh Chris W. , Adam Julie , Ratcliffe Peter J. 
Published: June 2022

Abstract
Clear cell renal cell carcinoma (ccRCC) is driven by biallelic inactivation of von Hippel Lindau (VHL) tumour suppressor gene. While ccRCC genesis has been associated with several signaling and metabolic pathways, the exact process by which VHL inactivation becomes tumorigenic remains unknown. We surmised that observing the behaviour and gene expression profiles of Vhl-inactivated cells in vivo with spatiotemporal resolution would help define cell types and conditions in which this genetic lesion is on balance tumorigenic. Here we report a novel lineage marking model of in vivo Vhl inactivation and describe the immediate consequences of Vhl loss in different nephron segments at cellular resolution. Studying Vhl inactivation with single-cell resolution requires a system in which Vhl-inactivated cells can be accurately identified. We developed a mouse model in which Vhl recombination is structurally linked to the expression of a tdTomato reporter cassette. This allows recombined cells to be visualised and isolated based on tdTomato expression. We coupled this model with the renal tubular epithelium (RTE) restricted Pax8-CreERT2 to induce biallelic (KO/KO) or monoallelic (wt/KO) inactivation of Vhl and observed the immediate fates of affected cells. Vhl is thought to be haplosufficient; thus, the wt/KO mice act as lineage-marked Vhl-competent controls to lineage-marked Vhl-null cells. We first observed the behavior of Vhl KO cells in the RTE over time. Vhl was inactivated by a controlled dose of tamoxifen that induced recombination in a subset of cells across the nephron. Three weeks after recombination, Vhl KO cells were morphologically normal but had stabilized HIF-2, confirming a reported ‘HIF-isoform’ switch. There were equal numbers of tdTomato-positive cells in the KO/KO and wt/KO kidneys, suggesting that initial recombination is equal between the genotypes and that there is no immediate distortion in cell fates following Vhl KO. However, four months after recombination, there were fewer tdTomato-positive cells in the KO/KO kidneys, suggesting a gradual removal or loss of Vhl KO from tissue. This was most prominent in the renal papilla and cortex. Additionally, some Vhl KO cells presented with morphological signs of cellular stress and altered differentiation. Taken together, we believe Vhl KO cells are subjected to profound selection in the nephron and that cell-type-specific, negative, and heterogenous consequences of Vhl KO impact on cell survival. One corollary is that many cells which ultimately survive in Vhl KO organs are in fact Vhl-competent. By intrinsically coupling Vhl-inactivation and lineage marking, our model allows these populations to be distinguished and followed over time. We are now studying the differential gene expression patterns of Vhl KO cells in cells of the RTE and aim to define genetic signatures for contexts in which Vhl KO cells survive and contribute to ccRCC genesis.
Citation Format: Joanna D C Lima, Samvid Kurlekar, Chris W. Pugh, Julie Adam, Peter J. Ratcliffe. A novel lineage-tracing model of Vhldeletion reveals time-dependent cellular changes in the kidney [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 939.