{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:57:37Z","timestamp":1782280657477,"version":"3.54.5"},"posted":{"date-parts":[[2025,3,12]]},"group-title":"Cancer Biology","reference-count":61,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T00:00:00Z","timestamp":1741737600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,22]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>\n                    NUT carcinoma is a rare but highly lethal solid tumor without an effective standard of care. NUT carcinoma is caused by bromodomain-containing\n                    <jats:italic>NUTM1<\/jats:italic>\n                    fusion oncogenes, most commonly\n                    <jats:italic>BRD4::NUTM1<\/jats:italic>\n                    . BRD4::NUTM1 recruits p300 to acetylate H3K27 forming expansive stretches of hyperacetylated chromatin called \u201cmegadomains\u201d with the overexpression of corresponding oncogenes, including\n                    <jats:italic>MYC<\/jats:italic>\n                    . We hypothesized that transcriptional dysregulation caused by BRD4::NUTM1 would lead to the generation of cancer-specific antigens that could be therapeutically actionable.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    We integrated genomics, computational antigen prediction software, targeted immunopeptidomics using single- and double-labeled peptide standards, and gain\/loss-of-function genetic experiments on a panel of cell lines (N=5), a patient derived xenograft, a tissue microarray (N=77), and patient samples from the Tempus AI Sequencing Database harboring evidence of\n                    <jats:italic>NUTM1<\/jats:italic>\n                    fusions (N=165). We created an \u03b1PRAME\n                    <jats:sub>425<\/jats:sub>\n                    T-cell receptor x SP34 \u03b1CD3 bispecific molecule modeled after brenetafusp, an \u03b1PRAME\n                    <jats:sub>425<\/jats:sub>\n                    T-cell receptor bispecific T-cell engager, as well as \u03b1PRAME\n                    <jats:sub>425<\/jats:sub>\n                    TCR T-cells based on anzutresgene autoleucel and we applied these products to NUT carcinoma cells\n                    <jats:italic>in vitro<\/jats:italic>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    We identified\n                    <jats:italic>PRAME<\/jats:italic>\n                    as the most commonly expressed cancer\/testis antigen in patient samples harboring the three canonical NUT carcinoma fusions (\n                    <jats:italic>BRD4::NUTM1<\/jats:italic>\n                    ,\n                    <jats:italic>BRD3::NUTM1<\/jats:italic>\n                    , and\n                    <jats:italic>NSD3::NUTM1<\/jats:italic>\n                    ). Additionally, 56% (43\/77) of NUT carcinoma tissue microarray samples stained positive for PRAME.\n                    <jats:italic>BRD4::NUTM1<\/jats:italic>\n                    expression in HEK 293T cells enhanced PRAME levels and\n                    <jats:italic>BRD4::NUTM1<\/jats:italic>\n                    knockout in NUT carcinoma cells reduced PRAME levels. Immunopeptidomics detected more PRAME-derived HLA ligands (N=9) than all other cancer\/testis antigens combined (N=5). Targeted mass spectrometry detected the HLA-A*02:01\/SLLQHLIGL (PRAME\n                    <jats:sub>425<\/jats:sub>\n                    ) epitope in 100% (4\/4) of HLA-A*02+, PRAME+ NUT carcinoma samples at higher levels (&gt;0.01 fM) than HLA-A*02:01\/RLDQLLRHV (PRAME\n                    <jats:sub>312<\/jats:sub>\n                    ) or HLA-A*02:01\/YLHARLREL (PRAME\n                    <jats:sub>462<\/jats:sub>\n                    ). The \u03b1PRAME\n                    <jats:sub>425<\/jats:sub>\n                    T-cell receptor x SP34 \u03b1CD3 bispecific molecule and \u03b1PRAME\n                    <jats:sub>425<\/jats:sub>\n                    TCR T-cells each exhibited potent, T-cell mediated cytotoxicity against\n                    <jats:italic>PRAME<\/jats:italic>\n                    + NUT carcinoma cells.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>\n                    <jats:italic>PRAME<\/jats:italic>\n                    is highly and frequently expressed in NUT carcinoma and the most common oncoprotein causing NUT carcinoma, BRD4::NUTM1, contributes to these high PRAME levels. PRAME epitopes presented by HLA Class I are a previously unrecognized therapeutic vulnerability for NUT carcinoma that warrant clinical trials testing PRAME targeted immunotherapies in this neglected patient population.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>What is already known on this topic<\/jats:title>\n                  <jats:p>NUT carcinoma is a devastating malignancy that is recalcitrant to cytotoxic chemotherapy, T-cell checkpoint blockade, and targeted therapies in the form of bromodomain inhibitors.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>What this study adds<\/jats:title>\n                  <jats:p>\n                    NUT carcinoma tumors are high in the cancer\/testis gene\n                    <jats:italic>PRAME<\/jats:italic>\n                    . The oncogene most commonly causing NUT carcinoma,\n                    <jats:italic>BRD4::NUTM1<\/jats:italic>\n                    , contributes to these high levels. NUT carcinoma cells present PRAME epitopes on HLA Class I molecules and are susceptible to PRAME-directed, T-cell mediated cytotoxicity.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>How this study might affect research, practice or policy<\/jats:title>\n                  <jats:p>\n                    Our results argue for phase I\/II clinical trials testing PRAME immunotherapies like brenetafusp or anzutresgene autoleucel in\n                    <jats:italic>PRAME<\/jats:italic>\n                    + NUT carcinoma patients.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"642090v3_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.07.642090","type":"posted-content","created":{"date-parts":[[2025,3,13]],"date-time":"2025-03-13T12:54:52Z","timestamp":1741870492000},"source":"Crossref","is-referenced-by-count":2,"title":["Shared PRAME Epitopes are T-Cell Targets in NUT Carcinoma"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8821-4980","authenticated-orcid":false,"given":"Jeffrey L.","family":"Jensen","sequence":"first","affiliation":[{"name":"Division of Medical Oncology, Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina"},{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-9858-2477","authenticated-orcid":false,"given":"Sara K.","family":"Peterson","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"},{"name":"Department of Genetics, Curriculum in Bioinformatics and Computational Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Sambade","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica R.","family":"Alley","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shawn","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoaki","family":"Kinjo","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"},{"name":"Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4605-2776","authenticated-orcid":false,"given":"Sarah N.","family":"Bennett","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"},{"name":"Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"II","given":"Steven P.","family":"Vensko","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North 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A.","family":"Price","sequence":"additional","affiliation":[{"name":"Tempus AI, Inc., Chicago Illinois"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kwangok P.","family":"Nickel","sequence":"additional","affiliation":[{"name":"Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, University of Wisconsin, Madison, WI"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Randall J.","family":"Kimple","sequence":"additional","affiliation":[{"name":"Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, University of Wisconsin, Madison, WI"},{"name":"University of Wisconsin Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, University of Wisconsin, Madison, WI"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1836-4075","authenticated-orcid":false,"given":"Rishi S.","family":"Kotecha","sequence":"additional","affiliation":[{"name":"Department of Clinical Haematology, Oncology, Blood and Marrow Transplantation, Perth Children\u2019s Hospital, Perth, Australia"},{"name":"Leukaemia Translational Research Laboratory, WA Kids Cancer Centre, The Kids Research Institute Australia, University of Western Australia, Perth, Australia"},{"name":"Curtin Medical School, Curtin University, Perth, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura E.","family":"Herring","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1552-0960","authenticated-orcid":false,"given":"Ian J.","family":"Davis","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina"},{"name":"Division of Hematology-Oncology, Department of 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