Abstract PR012: Co-operation of <i>vhl</i> and <i>pbrm1</i> in early ccRCC morphogenesis

Abstract Characterizing the early events preceding tumorigenesis is a major challenge in understanding cancer.A key insight emerging from this work is to understand the genesis of clear cell renal cell carcinoma (ccRCC) that is driven by two frequently observed sites of mutations, von Hippel Lindau (VHL) tumor suppressor gene and Polybromo-1 (PBRM1), a subunit of the PBAF SWI/SNF chromatin remodeling complex. Biallelic inactivating mutations in VHL are observed in at least 80% of ccRCC, whereas such mutations in PBRM1 are observed in approximately 40% of ccRCC,generally occurring after VHL inactivation. Concomitant Vhl and Pbrm1 deletion, but not Vhl deletion alone, in the kidney has been reported to be sufficient to drive the formation of renal tumors. However, the oncogenic synergetic interaction between VHL and PBRM1 inactivation remains poorly studied. We have previously reported an oncogenic cell labelling strategy in which Vhl is linked to tdTomato expression allowing accurate identification and retrieval of marked Vhl-null cells. Here, we have combined this system with conditional Pbrm1 deletion to compare transcriptional and morphological phenotypes in Vhl versus Vhl/Pbrm1-null cells with single-cell resolution in advance of overt tumor formation. In our system, concomitant Vhl and Pbrm1 loss led to the formation of tdTomato renal tumors exhibiting cell clearing, stained positively for HIF1 and HIF2A and did not express PBRM1. Further, we sought to study the earliest contributions of Vhl and/or Pbrm1 inactivation to gene expression and morphological changes to gain mechanistic effects in the pathogenesis of ccRCC.Our analyses reveal 4 main results: 1) By single-cell data, we previously reported the changes in Vhl inactivation solely were dominated, direct or indirect, by alterations in the HIF. Here, we found surprisingly little effect of Pbrm1 inactivation on the HIF transcriptional response. 2) Previously we reported an early proliferative drive in Vhl that was abrogated over time. Here, this increase in proliferation was present in Vhl/Pbrm1 cells but it was attenuated compared with Vhl alone, however this proliferation was strikingly more sustained. 3)The distinct effect of pbrm1 inactivation was mainly involved with epithelial integrity, cell motility and lipid metabolism. 4) Finally, we captured the first morphological abnormalities in Vhl/Pbrm1 cells showing loss of epithelial identity and integrity, such as cells that had escaped the tubular structure, either entering the lumen or protruding through the basement membrane into the interstitium, also forming crowding and multilayer clusters stained for tdTomato with visible loss of brush border.We further confirmed these abnormal cells were proliferating by incorporation of BrdU even at earlier stages.Our findings suggest that through an early and Vhl-independent action of Pbrm1 inactivation disrupts epithelial organization allowing cells to breach the tubular structure, form multi-layered epithelia, retain proliferative capacity and eventually form renal tumors. Citation Format: Joanna Lima, Samvid Kurlekar, Norma Masson, Christopher Pugh, Julie Adam, David Mole, Peter Ratcliffe. Co-operation of vhl and pbrm1 in early ccRCC morphogenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr PR012.