WES analysis for all mouse SCCs and UVB-treated samples.

Opal and CODEX multiplexed tissue imaging data shows infiltration of proliferative CD8+ T-cells to the rejected SCCs.

Differentially expressed genes in tumor compartment at days 0, 6, 10, and 16 post-inoculation.

Differential expression analysis of rejected vs. non-rejected tumor cells.

List of antibodies used for IHC staining of human samples.

Transcriptional changes in the Mif KO compared to Mif WT tumors.

DNA mutations are a well-characterized source of neoepitopes in immunotherapy. Here, we examined the contribution of dysregulated RNA processing to neoantigen production. Leveraging multi-omics and checkpoint inhibitor (CPI) response data from >1,000 patients, we identified reduced activity of the nonsense-mediated mRNA decay (NMD) pathway kinase SMG1 as a predictor of improved CPI response. NMD inhibition through SMG1 targeting stabilized transcripts containing premature termination codons, most of which were of non-mutational origin. This reshaped the major histocompatibility complex class I (MHC class I)-bound immunopeptidome and increased neoantigen abundance to levels comparable to high mutation burden tumors. Functionally, NMD inhibition drove antigen-dependent T cell-mediated tumor cell killing in vitro, promoted activation of tissue-resident T cells in patient-derived models ex vivo, and improved CPI efficacy in vivo. Our findings establish NMD inhibition as a strategy to harness a previously inaccessible source of canonical and non-canonical neoantigens, with the potential to increase tumor immunogenicity across cancers.

Supplementary data linked to the in vivo CRISPR screen.

Clinical data and characterization of the single-cell clones.

CellChat and CellPhoneDB analyses reveal increased MIF signaling from tumor cells to macrophages in non-rejected clones.

Immunopeptidomics and TCR sequencing analyses showed similar results for the rejected and non-rejected SCCs.

New approaches that generate long-lasting therapeutic responses in patients with therapy-resistant metastatic cancer are urgently needed. To address this challenge, we developed Spot Neoantigens in Metastases (SpotNeoMet), a novel data-driven pipeline that systematically identifies recurrently presented neopeptides in treatment-resistant patients. We identified seven therapy resistance mutations predicted to produce neopeptides presented by common HLAs. Using HLA immunopeptidomics, we discovered three novel neopeptides derived from androgen receptor (AR) H875Y, a common metastatic castration-resistant prostate cancer (mCRPC) mutation. We validated these neoantigens as highly immunogenic and then isolated and characterized cognate T-cell receptors (TCR) from healthy donor peripheral blood mononuclear cells. We demonstrated that AR H875Y-specific TCRs are highly specific and kill prostate cancer cells presenting AR neopeptides in vitro and in vivo. Our new pipeline identifies novel immunotherapy targets and potential treatment options for patients with mCRPC. Moreover, SpotNeoMet offers a systematic route to identify "HLA-peptide" pairs and their cognate TCRs across treatment-resistant cancers. SIGNIFICANCE:As the emergence of resistance to targeted treatments in patients with metastatic cancer, there is an urgent need for innovative therapeutic approaches for this population. Our study provides a new analytic framework to identify neoantigens from treatment-resistant mutations and a proof-of-concept T cell-based immunotherapy treatment for mCRPC.

Protein synthesis is tightly regulated in cells; however, in cancer, ribosomes deviate from canonical translation, generating altered protein products. These deviations arise from cell-intrinsic alterations, as well as extrinsic pressures within the tumor microenvironment, collectively reshaping the translational landscape and reducing translation fidelity. Translational recoding in cancer expands proteome diversity and promotes tumor fitness by enhancing stress adaptation, metabolic, and phenotypic plasticity. At the same time, recoding events generate peptides that are often presented as tumor-specific antigens, thereby eliciting immune responses against cancer. Accordingly, therapeutic strategies that modulate translational fidelity and induce recoding are emerging to enhance tumor immunogenicity and improve immunotherapy responses. Here, we examine the drivers and consequences of translational recoding in cancer, its dual role in promoting tumor adaptation while shaping immune surveillance, and its potential as a targetable vulnerability in cancer therapy.

Evaluation of Mif expression in stromal cells, and its effect on patient survival.

Cell types from scRNA-seq of tumors from mice inoculated with rejected or non-rejected SCCs.

scRNA-seq characterization of Mif KO tumor clones.

List of antibodies used for CODEX multiplexed tissue imaging

List of primers used for sgRNA library generation

CyTOF analysis comparing tumors derived from Mif WT and Mif KO validating the scRNAseq findings.

CD8+ T cell depletion data. Supplemental figure to Figure 6F.