Year: 2026
Type: preprint
Abstract: 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. Highlights Fusion proteins activate in- and out-of-frame neoproteins through intragenic transcription. Neoproteins exhibit distinct protein structures and localizations compared to their host genes. Mass spectrometry detects 911 cryptic proteins, including 29 EwS neoproteins, 15 HLA-I presented. TRPM4-dORF is a stop-less 80-aa neoprotein with abundant peptide evidence exclusively in EwS.
Authors: Luuk A. Broeils, Jill Pilet, Mariia K. Koshkina, Jing Li, Sandrine Grossetete, Emma G. A. Westerink, Orson Jay, Lotta Smit, Ting Luo, Erkan Narmanli, Ana Pinheiro Lopes, Florian H. Geyer, Sem A. G. Engels, Clémence Henon, Karim Aljakouch, Jeroen Krijgsveld, Ismail Jamail, Sylvain Baulande, Sonia Lameiras, Martha J. Carreño-Gonzalez, Georg W. Omlor, Jonathan M. Mudge, Valerie U. Nguyen, Michal I. Swirski, Hakon Tjeldnes, Eivind Valen, Burkhard Lehner, Uta Dirksen, Wolfgang Faigle, Ana I. Lalanne, Olivier Lantz, Karine Laud-Duval, Christina Michail, Michael VanInsberghe, Alexander van Oudenaarden, Nina Hahnen, Nadine Gmelin, Johannes H. M. Merks, Ferhat Alkan, Joshua J. Waterfall, Thomas G. P. Grünewald, Olivier Delattre, Sebastiaan van Heesch
Institutions: Oncode Institute, Princess Máxima Center, Inserm, Université Paris Sciences et Lettres, Institut Curie, German Cancer Research Center, University Hospital Heidelberg, National Center for Tumor Diseases, Hopp Children's Cancer Center Heidelberg, Deutsches Konsortium für Translationale Krebsforschung, Heidelberg University, European Bioinformatics Institute, University of Oslo, Essen University Hospital, Royal Netherlands Academy of Arts and Sciences, University Medical Center Utrecht, Utrecht University
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
Funders: Cancer Research Institute, St. Baldrick's Foundation, Deutsches Krebsforschungszentrum, Equipex, Cancer Research UK, European Commission, Agence Nationale de la Recherche, Institut National de la Santé et de la Recherche Médicale, Bundesministerium für Bildung und Forschung, Bundesministerium für Gesundheit, Nederlandse Organisatie voor Wetenschappelijk Onderzoek, Ligue Contre le Cancer, Deutsche Krebshilfe, Stichting Kinderen Kankervrij, Institut National Du Cancer, Deutsche Kinderkrebsstiftung, Direction Générale de l’offre de Soins, Deutschen Konsortium für Translationale Krebsforschung, Oncode Institute, CRIS Cancer Foundation, HORIZON EUROPE Framework Programme, National Cancer Institute, H2020 Marie Skłodowska-Curie Actions, Eurostars
Awards: Smart Objects For Intelligent Applications, G1554707310, G1625433374, G2579310621, G2580753410, G271172329, G315159858, G4072984709, G4463545759, G449165211, G5125889110, G5467192712, G6062526893, G6864960027, G705061367, Netherlands X-omics Initiative, G786270660, G7999552054, Verbund HEROES-AYA - Heterogenität, Evolution und Resistenz von durch Fusionsgene getriebenen Sarkomen bei Jugendlichen und jungen Erwachsenen, Standort Universität Duisburg-Essen
OpenAlex ID: W7213516622