L. A. Broeils, J. Pilet, M. K. Koshkina, J. Li, S. Grossetete, E. G. A. Westerink, O. Jay, L. Smit, T. Luo, E. Narmanli, A. P. Pinheiro Lopes, F. H. Geyer, S. A. G. Engels, C. Henon, K. Aljakouch, J. Krijgsveld, I. Jamail, S. Baulande, S. Lameiras, M. J. Carreno-Gonzalez, G. W. Omlor, J. M. Mudge, V. U. Nguyen, M. I. Swirski, H. Tjeldnes, E. Valen, B. Lehner, U. Dirksen, W. Faigle, A. I. Lalanne, O. Lantz, K. Laud-Duval, C. Michail, M. VanInsberghe, A. van Oudenaarden, N. Hahnen, N. Gmelin, J. H. M. Merks, F. Alkan, J. J. Waterfall, T. G. P. Gruenewald
How tumors generate a cryptic "dark" proteome absent from healthy tissues, and whether it can be reversibly activated, remains unclear. In Ewing sarcoma, all tumors are driven by EWSR1::ETS fusions, making it a tractable model to study dark proteome activation. Integrating matched transcriptomes, translatomes and proteomes from 48 patient tumors with long-read RNA sequencing, single-cell Ribo-seq, proteomics and immunopeptidomics in cell line models, we show that EWSR1::FLI1 acts as a reversible switch recurrently inducing hundreds of cancer-specific neoproteins. Many arise from canonical coding regions via intragenic transcription start sites, generating truncated or out-of-frame neoproteins. We uncover a fusion-dependent increase in ribosomal readthrough into poly(A) tails, generating a stable 80-amino-acid TRPM4 neoprotein that, despite lacking a stop codon, is the most abundant tumor-specific microprotein across patients. Proteomic, immunopeptidomic, and immunofluorescence analyses validate neoproteins as tumor-restricted antigens, revealing a therapeutically actionable cancer dark proteome controlled by one oncogenic fusion.