Oncogenic fusions induce an extensive cancer-restricted cryptic proteome in Ewing sarcoma

Broeils, L. A.; Pilet, J.; Koshkina, M. K.; Li, J.; Grossetete, S.; Westerink, E. G. A.; Jay, O.; Smit, L.; Luo, T.; Narmanli, E.; Pinheiro Lopes, A. P.; Geyer, F. H.; Engels, S. A. G.; Henon, C.; Aljakouch, K.; Krijgsveld, J.; Jamail, I.; Baulande, S.; Lameiras, S.; Carreno-Gonzalez, M. J.; Omlor, G. W.; Mudge, J. M.; Nguyen, V. U.; Swirski, M. I.; Tjeldnes, H.; Valen, E.; Lehner, B.; Dirksen, U.; Faigle, W.; Lalanne, A. I.; Lantz, O.; Laud-Duval, K.; Michail, C.; VanInsberghe, M.; van Oudenaarden, A.; Hahnen, N.; Gmelin, N.; Merks, J. H. M.; Alkan, F.; Waterfall, J. J.; Gruenewald, T. G. P.

doi:10.64898/2026.09.16.751792

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bioRxiv · 10.64898/2026.09.16.751792

Oncogenic fusions induce an extensive cancer-restricted cryptic proteome in Ewing sarcoma

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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.

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Broeils, L. A., Pilet, J., Koshkina, M. K., Li, J., Grossetete, S., Westerink, E. G. A., Jay, O., Smit, L., Luo, T., Narmanli, E., Pinheiro Lopes, A. P., Geyer, F. H., Engels, S. A. G., Henon, C., Aljakouch, K., Krijgsveld, J., Jamail, I., Baulande, S., Lameiras, S., Carreno-Gonzalez, M. J., Omlor, G. W., Mudge, J. M., Nguyen, V. U., Swirski, M. I., Tjeldnes, H., Valen, E., Lehner, B., Dirksen, U., Faigle, W., Lalanne, A. I., Lantz, O., Laud-Duval, K., Michail, C., VanInsberghe, M., van Oudenaarden, A., Hahnen, N., Gmelin, N., Merks, J. H. M., Alkan, F., Waterfall, J. J., Gruenewald, T. G. P.. 2026-09-17. Oncogenic fusions induce an extensive cancer-restricted cryptic proteome in Ewing sarcoma. https://doi.org/10.64898/2026.09.16.751792

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