Abstract 3024: HIF1A and HIF2A differentially contribute to early and adaptive changes following in vivo Vhl inactivation in the kidney

Abstract The von Hippel Lindau (VHL) tumor suppressor gene is a classical tumor suppressor whose biallelic inactivation is a truncal event in clear cell renal cell carcinoma (ccRCC) evolution. VHL is best characterized as the recognition component of an E3 ubiquitin ligase complex that targets hypoxia inducible transcription factors (HIF1A and HIF2A) for conditional oxygen-dependent degradation. HIF activation following VHL inactivation is an early event in ccRCC progression. While the functions of HIF1A and HIF2A in ccRCC biology and progression have been studied, those in VHL-null non-neoplastic cells of the renal tubular epithelium (RTE) and their contribution to tumorigenesis have not been fully explored. Here, using a combination of lineage marking and single-cell RNA sequencing (scRNA-seq), we provide the first description of HIF1A- and/or HIF2A-dependent and -independent consequences of in vivo Vhl inactivation in the RTE with single-cell resolution. We have previously reported on the development of a novel mouse model designed to mark and track cells that have undergone Vhl inactivation in vivo. Using this model, we have shown that Vhl inactivation has extensive, cell-type specific effects on gene expression in cells of the RTE within 3 weeks of recombination. Furthermore, we have shown that over a course of 4-8 months after Vhl inactivation, Vhl-null cells undergo specific adaptive changes in gene expression that encompass genes not regulated early after Vhl inactivation. In the work reported here, we have coupled this mouse model to a tamoxifen-inducible RTE-specific Pax8-CreERT2 and conditional alleles for Hif1a and/or Epas1 (codes for HIF2A). We have analyzed ‘marked’ recombined cells at early (1-3 weeks) or late (4-8 months) timepoints after recombination and compared these cells to time-matched Vhl-null cells using immunohistochemistry, in situ RNA hybridization and scRNA-seq. We found that though both HIF isoforms were present in Vhl-null cells at the early timepoint, expression of genes regulated early after Vhl inactivation was affected more by Hif1a inactivation than Epas1 inactivation. In contrast, expression of genes that are regulated in Vhl-null cells over time was drastically impacted by Epas1 deletion but was unaffected by Hif1a deletion. Interestingly, most of these Epas1-dependent, time-dependent changes in Vhl-null cells involved genes that have not been identified as direct transcriptional targets of HIF. Collectively, our work suggests that HIF1A and HIF2A have temporally distinct contributions to gene expression in Vhl-null cells in the RTE. We are currently analyzing the cell-type specificity of these contributions and ascertaining their relevance to the adoption of ccRCC-associated gene expression profiles. Citation Format: Samvid Kurlekar, Joanna Lima, Ayslan B. Barros, Norma Masson, Sophia Zhai, Christopher W. Pugh, Julie Adam, Peter J. Ratcliffe. HIF1A and HIF2A differentially contribute to early and adaptive changes following in vivo Vhl inactivation in the kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3024.