Abstract The tumor cell surface proteome (“surfaceome”) plays a pivotal role in mediating cancer-microenvironment interactions and represents a rich source of therapeutic and diagnostic targets. In clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, loss of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark and primary driver, which subsequently leads to hypoxia-inducible factor 2α (HIF2α) activation. Comprehensive mapping of the ccRCC surfaceome zhas been limited by technical constraints, leaving many clinically relevant membrane proteins undiscovered. To systematically identify membrane-bound pVHL/HIF2α-regulated proteins, we employed a peroxidase-mediated proximity biotinylation proteomics strategy using horseradish peroxidase (HRP) fused to the transmembrane domain of PDGFR and a membrane-impermeable substrate, biotin-xx-phenol (BxxP), to selectively label cell-surface proteins. Biotinylated proteins were enriched with streptavidin beads, digested on-bead, and analyzed using tandem mass tag (TMT) -based quantitative mass spectrometry (MS). Using 786-O and OSRC2 ccRCC VHL -/- cell lines engineered to re-express VHL in a DOX-inducible manner, we profiled surfaceome changes upon DOX, HIF2a inhibitor PT2399 or DMSO treatment. Known HIF2-regulated membrane proteins (e. g. , GLUT1, NDRG1) were recovered, validating the approach. We further identified and validated SEMA5B as a novel pVHL/HIF2-responsive membrane protein, elevated in ccRCC tumors relative to normal kidney (TCGA datasets and in patients). Importantly, SEMA5B is absent or lowly expressed in most vital organs. Additionally, Orthotopic xenograft assays demonstrated that SEMA5B loss impaired tumor growth, which was rescued by exogenous re-expression, indicating its essential role in ccRCC tumorigenic activity in vivo. Our study establishes oncogene-driven surfaceome profiling as a robust strategy to uncover cancer-specific vulnerabilities and highlights SEMA5B as a potential therapeutic membrane target in ccRCC, paving the way for the development of precision-targeted therapies and immunotherapies that exploit the unique surface landscape of cancer cells. Citation Format: Yanyan Hu, Khanh Nguyen, Namrata Udeshi, Berkay Simsek, Gabriel Roberti De Oliveira, Maxwell Darrow Seager, Jiefu Li, Signoretti Sabina, Steven A. Carr, William G. Kaelin. Profiling the ccRCC surfaceome reveals SEMA5B as a potential therapeutic membrane target 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 (5Suppl₂): Abstract nr B018.
Hu et al. (Fri,) studied this question.