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Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration. Fusion proteins in cancers with low mutational burden represent functional neoantigens that elicit T cell activation and mediate responses to immunotherapy.

Purpose: Salivary gland carcinomas (SGC) are rare, aggressive cancers with high rates of recurrence and distant metastasis. These factors, and a lack of active systemic therapies, contribute to poor clinical outcome. Response rates with immune checkpoint blockade have been low, although clinical data remain sparse. To improve the efficacy of therapies, a more comprehensive understanding of relevant molecular alterations and immunologic processes is needed. Experimental Design: To characterize the immune microenvironment and neoantigen landscape of SGCs, we performed RNA sequencing (RNA-seq) in 76 tumors representing the three most lethal histologies: adenoid cystic carcinoma (ACC), myoepithelial carcinoma (MECA), and salivary duct carcinoma (SDC). We analyzed transcriptomic profiles, tumor-infiltrating immune cell populations, and measures of T-cell activation/dysfunction. In 37 cases also undergoing exome sequencing, we analyzed somatic mutations and neoantigens. Results: SDCs exhibited high levels of immune infiltration, with corresponding higher levels of T-cell dysfunction, and higher mutational load. In contrast, ACCs were characterized by an immune-excluded microenvironment, the presence of M2-polarized macrophages and myeloid-derived suppressor cells, and very low mutational load. MECAs were more heterogeneous, with both immune-low and immune-high phenotypes represented. Across all SGCs, levels of immune infiltration were associated with mutation- and fusion-derived neoantigens, and with aggressive clinical behavior. Conclusions: These findings provide new insights into the immune microenvironment and neoantigen landscape of SGCs, showing that mechanisms of immune escape appear to differ by histology. These data nominate potential immunologic vulnerabilities and may help guide the next steps of investigation in precision immunotherapy for these difficult-to-treat cancers.

Nucleolin (NCL) is an abundant stress-responsive, RNA-binding phosphoprotein that controls gene expression by regulating either mRNA stability and/or translation. NCL binds to the AU-rich element (ARE) in the 3'UTR of target mRNAs, mediates miRNA functions in the nearby target sequences, and regulates mRNA deadenylation. However, the mechanism by which NCL phosphorylation affects these functions and the identity of the deadenylase involved, remain largely unexplored. Earlier we demonstrated that NCL phosphorylation is vital for cell cycle progression and proliferation, whereas phosphorylation-deficient NCL at six consensus CK2 sites confers dominant-negative effect on proliferation by increasing p53 expression, possibly mimicking cellular DNA damage conditions. In this study, we show that NCL phosphorylation at those CK2 consensus sites in the N-terminus is necessary to induce deadenylation upon oncogenic stimuli and UV stress. NCL-WT, but not hypophosphorylated NCL-6/S*A, activates poly (A)-specific ribonuclease (PARN) deadenylase activity. We further demonstrate that NCL interacts directly with PARN, and under non-stress conditions also forms (a) complex (es) with factors that regulate deadenylation, such as p53 and the ARE-binding protein HuR. Upon UV stress, the interaction of hypophosphorylated NCL-6/S*A with these proteins is favored. As an RNA-binding protein, NCL interacts with PARN deadenylase substrates such as TP53 and BCL2 mRNAs, playing a role in their downregulation under non-stress conditions. For the first time, we show that NCL phosphorylation offers specificity to its protein-protein, protein-RNA interactions, resulting in the PARN deadenylase regulation, and hence gene expression, during cellular stress responses.

Myoepithelial carcinoma (MECA) is an aggressive salivary gland cancer with largely unknown genetic features. Here we comprehensively analyze molecular alterations in 40 MECAs using integrated genomic analyses. We identify a low mutational load, and high prevalence (70%) of oncogenic gene fusions. Most fusions involve the PLAG1 oncogene, which is associated with PLAG1 overexpression. We find FGFR1-PLAG1 in seven (18%) cases, and the novel TGFBR3-PLAG1 fusion in six (15%) cases. TGFBR3-PLAG1 promotes a tumorigenic phenotype in vitro, and is absent in 723 other salivary gland tumors. Other novel PLAG1 fusions include ND4-PLAG1; a fusion between mitochondrial and nuclear DNA. We also identify higher number of copy number alterations as a risk factor for recurrence, independent of tumor stage at diagnosis. Our findings indicate that MECA is a fusion-driven disease, nominate TGFBR3-PLAG1 as a hallmark of MECA, and provide a framework for future diagnostic and therapeutic research in this lethal cancer., Myoepithelial carcinoma (MECA) is a rare aggressive salivary gland cancer. Here, the authors analyze the genomic landscape of MECA and identify a high prevalence of oncogenic gene fusions, primarily PLAG1 fusions, highlighting TGFBR3-PLAG1 as a potential hallmark of MECA.

Myoepithelial carcinoma (MECA) is an aggressive type of salivary gland cancer with largely unknown molecular features. MECA may arise de novo or result from oncogenic transformation of a pre-existing pleomorphic adenoma (MECA ex-PA). We comprehensively analyzed the molecular alterations in MECA with integrated genomic analyses. We identified a low mutational load (0.5/MB), but a high prevalence of fusion oncogenes (28/40 tumors; 70%). We foundFGFR1-PLAG1in 7 (18%) cases, and the novelTGFBR3-PLAG1fusion in 6 (15%) cases.TGFBR3-PLAG1was specific for MECA de novo tumors or the malignant component of MECA ex-PA, was absent in 723 other salivary gland tumors, and promoted a tumorigenic phenotype in vitro. We discovered other novelPLAG1fusions, includingND4-PLAG1,which is an oncogenic fusion between mitochondrial and nuclear DNA. One tumor harbored anMSN-ALKfusion, which was tumorigenic in vitro, and targetable with ALK inhibitors. Certain gene fusions were predicted to result in neoantigens with high MHC binding affinity. A high number of copy number alterations was associated with poorer prognosis. Our findings indicate that MECA is a fusion-driven disease, nominateTGFBR3-PLAG1as a hallmark of MECA, and provide a framework for future steps of diagnostic and therapeutic research in this lethal cancer.

Nucleolin (NCL) is a major nucleolar phosphoprotein that has pleiotropic effects on cell proliferation and is elevated in a variety of tumors. NCL is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (CDK1). Earlier we demonstrated that a NCL-mutant that is partly defective in undergoing phosphorylation by CK2 inhibits chromosomal replication through its interactions with Replication Protein A, mimicking the cellular response to DNA damage. We further delineated that the N-terminus of NCL associates with Hdm2, the most common E3 ubiquitin ligase of p53. We reported that NCL antagonizes Hdm2 to stabilize p53 and stimulates p53 transcriptional activity. Although NCL-phosphorylation by CK2 and ribosomal DNA transcription are closely coordinated during interphase, the role of NCL phosphorylation in regulating cell proliferation remains unexplored. We have therefore engineered unique human cells that specifically induce expression of NCL-wild type (WT) or a phosphorylation-deficient NCL-mutant, 6/S*A where all the six CK2 consensus serine sites residing in the N-terminus NCL were mutated to alanine. Here we show that this NCL-mutant is defective in undergoing phosphorylation by CK2. We also demonstrate that NCL-phosphorylation by CK2 is required through the S-phase progression in cell cycle and hence proliferation. Induced expression of NCL with mutated CK2 phosphorylation sites stabilizes p53, results in higher expression of Bcl2 (B-cell lymphoma 2) homology 3 (BH3)-only apoptotic markers and causes a dominant-negative effect on cell viability. Our unique cellular system thus provides the first evidential support to delineate phospho-specific functions of NCL on cell proliferation.