bioRxiv : the preprint server for biology · 2025
Shared PRAME Epitopes are T-Cell Targets in NUT Carcinoma.
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Jensen Jeffrey L, Peterson Sara K, Sambade Maria, Alley Jessica R, Yu Shawn, Kinjo Tomoaki, Bennett Sarah N, Vensko Steven P, Shabrang Mitra, DeBetta Johnathan D, Geyer Julie K, Price Brandon A, Nickel Kwangok P, Kimple Randall J, Kotecha Rishi S, Herring Laura E, Davis Ian J, Wang Jeremy R, French Christopher A, Kuhlman Brian, Weiss Jared, Rubinsteyn Alex, Vincent Benjamin G
Аннотация
BACKGROUND: NUT carcinoma is a rare but highly lethal solid tumor without an effective standard of care. NUT carcinoma is caused by bromodomain-containing NUTM1 fusion oncogenes, most commonly BRD4::NUTM1 . BRD4::NUTM1 recruits p300 to acetylate H3K27 forming expansive stretches of hyperacetylated chromatin called "megadomains" with the overexpression of corresponding oncogenes, including MYC . We hypothesized that transcriptional dysregulation caused by BRD4::NUTM1 would lead to the generation of cancer-specific antigens that could be therapeutically actionable.
METHODS: 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 NUTM1 fusions (N=165). We created an αPRAME 425 T-cell receptor x SP34 αCD3 bispecific molecule modeled after brenetafusp, an αPRAME 425 T-cell receptor bispecific T-cell engager, as well as αPRAME 425 TCR T-cells based on anzutresgene autoleucel and we applied these products to NUT carcinoma cells in vitro .
RESULTS: We identified PRAME as the most commonly expressed cancer/testis antigen in patient samples harboring the three canonical NUT carcinoma fusions ( BRD4::NUTM1 , BRD3::NUTM1 , and NSD3::NUTM1 ). Additionally, 56% (43/77) of NUT carcinoma tissue microarray samples stained positive for PRAME. BRD4::NUTM1 expression in HEK 293T cells enhanced PRAME levels and BRD4::NUTM1 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 425 ) epitope in 100% (4/4) of HLA-A*02+, PRAME+ NUT carcinoma samples at higher levels (>0.01 fM) than HLA-A*02:01/RLDQLLRHV (PRAME 312 ) or HLA-A*02:01/YLHARLREL (PRAME 462 ). The αPRAME 425 T-cell receptor x SP34 αCD3 bispecific molecule and αPRAME 425 TCR T-cells each exhibited potent, T-cell mediated cytotoxicity against PRAME + NUT carcinoma cells.
CONCLUSIONS: PRAME 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.
WHAT IS ALREADY KNOWN ON THIS TOPIC: 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.
WHAT THIS STUDY ADDS: NUT carcinoma tumors are high in the cancer/testis gene PRAME . The oncogene most commonly causing NUT carcinoma, BRD4::NUTM1 , 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.
HOW THIS STUDY MIGHT AFFECT RESEARCH PRACTICE OR POLICY: Our results argue for phase I/II clinical trials testing PRAME immunotherapies like brenetafusp or anzutresgene autoleucel in PRAME + NUT carcinoma patients.
METHODS: 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 NUTM1 fusions (N=165). We created an αPRAME 425 T-cell receptor x SP34 αCD3 bispecific molecule modeled after brenetafusp, an αPRAME 425 T-cell receptor bispecific T-cell engager, as well as αPRAME 425 TCR T-cells based on anzutresgene autoleucel and we applied these products to NUT carcinoma cells in vitro .
RESULTS: We identified PRAME as the most commonly expressed cancer/testis antigen in patient samples harboring the three canonical NUT carcinoma fusions ( BRD4::NUTM1 , BRD3::NUTM1 , and NSD3::NUTM1 ). Additionally, 56% (43/77) of NUT carcinoma tissue microarray samples stained positive for PRAME. BRD4::NUTM1 expression in HEK 293T cells enhanced PRAME levels and BRD4::NUTM1 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 425 ) epitope in 100% (4/4) of HLA-A*02+, PRAME+ NUT carcinoma samples at higher levels (>0.01 fM) than HLA-A*02:01/RLDQLLRHV (PRAME 312 ) or HLA-A*02:01/YLHARLREL (PRAME 462 ). The αPRAME 425 T-cell receptor x SP34 αCD3 bispecific molecule and αPRAME 425 TCR T-cells each exhibited potent, T-cell mediated cytotoxicity against PRAME + NUT carcinoma cells.
CONCLUSIONS: PRAME 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.
WHAT IS ALREADY KNOWN ON THIS TOPIC: 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.
WHAT THIS STUDY ADDS: NUT carcinoma tumors are high in the cancer/testis gene PRAME . The oncogene most commonly causing NUT carcinoma, BRD4::NUTM1 , 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.
HOW THIS STUDY MIGHT AFFECT RESEARCH PRACTICE OR POLICY: Our results argue for phase I/II clinical trials testing PRAME immunotherapies like brenetafusp or anzutresgene autoleucel in PRAME + NUT carcinoma patients.