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Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin‐dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor‐suppressor proteins p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)‐specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA‐I) molecules was enhanced upon p14ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non‐canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA‐specific T cell responses by decreasing both absolute and relative MDA‐peptide presentation in melanoma.

BackgroundThe immunopeptidome is constantly monitored by T cells to detect foreign or aberrant HLA peptides. It is highly dynamic and reflects the current cellular state, enabling the immune system to recognize abnormal cellular conditions, such as those present in cancer cells. To precisely determine how changes in cellular processes, such as those induced by drug treatment, affect the immunopeptidome, quantitative immunopeptidomics approaches are essential.MethodsTo meet this need, we developed a pulsed SILAC-based method for quantitative immunopeptidomics. Metabolic labeling with lysine, arginine, and leucine enabled isotopic labeling of nearly all HLA peptides across all allotypes (> 90% on average). We established a data analysis workflow that integrates the de novo sequencing-based tool Peptide-PRISM for comprehensive HLA peptide identification with MaxQuant for accurate quantification.ResultsWe employed this strategy to explore the modulation of the immunopeptidome upon MAPK pathway inhibition (MAPKi) and to investigate alterations associated with early cellular responses to inhibitor treatment and acquired resistance to MAPKi. Our analyses demonstrated significant changes in the immunopeptidome early during MAPKi treatment and in the resistant state. Moreover, we identified putative tumor-specific cryptic HLA peptides linked to these processes that might represent exploitable targets for cancer immunotherapy.ConclusionsWe have developed a new mass spectrometric approach that allowed us to investigate the effects of common MAPK inhibitors on the immunopeptidome of melanoma cells. This finally led to the discovery of new potential targets for cancer immunotherapy.