Alterations in HLA Class I-Presented Immunopeptidome and Class I-Interactome upon Osimertinib Resistance in EGFR Mutant Lung Adenocarcinoma

SIMPLE SUMMARY: We sought to identify molecular mechanisms of lower efficacy of immunotherapy in epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma and the differences in those mechanisms with the emergence of tyrosine kinase inhibitor (TKI)-resistance. To this end, we conducted affinity purification and quantitative mass spectrometry-based proteomic profiling of human leukocyte antigen (HLA) Class I-presented immunopeptides and Class I-interacting proteins. This large-scale dataset revealed that the Class I-presented immunopeptidome was suppressed in two third-generation EGFR TKI, osimertinib-resistant lung adenocarcinoma cell lines compared to their isogenic TKI-sensitive counterparts. The whole-cell proteomic profiling show that antigen presentation complex proteins and immunoproteasome were downregulated upon EGFR TKI resistance. Furthermore, HLA class I-interactome profiling demonstrated altered interaction with key apoptosis and autophagy pathway proteins. In summary, our comprehensive multi-proteomic characterization in antigen presentation machinery provides potentially novel evidence of poor immune response in osimertinib-resistant lung adenocarcinoma. ABSTRACT: Immune checkpoint inhibitor (ICI) therapy has been a paradigm shift in the treatment of cancer. ICI therapy results in durable responses and survival benefit for a large number of tumor types. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has shown great efficacy treating EGFR mutant lung cancers; however, all patients eventually develop resistance. ICI therapy has not benefitted EGFR mutant lung cancer. Herein, we employed stable isotope labeling by amino acids in cell culture (SILAC) quantitative mass spectrometry-based proteomics to investigate potential immune escape molecular mechanisms in osimertinib resistant EGFR mutant lung adenocarcinoma by interrogating the alterations in the human leukocyte antigen (HLA) Class I-presented immunopeptidome, Class I-interactome, and the whole cell proteome between isogenic osimertinib-sensitive and -resistant human lung adenocarcinoma cells. Our study demonstrates an overall reduction in HLA class I-presented immunopeptidome and downregulation of antigen presentation core complex (e.g., TAP1 and ERAP1/2) and immunoproteasome in osimertinib resistant lung adenocarcinoma cells. Several key components in autophagy pathway are differentially altered. S100 proteins and SLC3A2 may play critical roles in reduced antigen presentation. Our dataset also includes ~1000 novel HLA class I interaction partners and hundreds of Class I-presented immunopeptides in EGFR mutant lung adenocarcinoma. This large-scale unbiased proteomics study provides novel insights and potential mechanisms of immune evasion of EGFR mutant lung adenocarcinoma.