Abstract

Background and purpose Lung adenocarcinoma (LUAD) has limited therapeutic targets and poor survival. Noncanonical open reading frames (ORFs) in long noncoding RNAs (lncRNAs) may encode functional microproteins, but their roles in LUAD remain unclear. This study aims to characterise the LINC00973-encoded microprotein L3EMP and to investigate its tumour-promoting mechanisms and therapeutic potential. Methods L3EMP was identified through ribosome profiling and RNA sequencing, with its expression confirmed by mass spectrometry (MS) and Western blotting. Its functional relevance was further validated using CRISPR/Cas9-mediated gene manipulation. Functional experiments were performed in LUAD cells and chimeric antigen receptor T (CAR-T) cell models to elucidate the underlying molecular mechanisms. The interaction between USP22 and SIRT1 was investigated via ubiquitination assays and signalling pathway profiling. Finally, immunotherapeutic potential was evaluated using synthetic L3EMP peptides and B7-H3-targeted CAR-T cells. Key results (1) L3EMP, a microprotein encoded by LINC00973, is overexpressed in LUAD and its expression level is correlated with poor prognosis. (2) L3EMP stabilises SIRT1 by promoting USP22-mediated deubiquitination. This forms a positive feedback loop involving YY1 that activates AKT/ERK signalling, thereby promoting proliferation and invasion. (3) L3EMP knockout suppresses tumour growth in PDX models. (4) Synthetic L3EMP peptide enhances antitumor immunity as a neoantigen. (5) L3EMP deletion synergises with B7-H3 CAR-T therapy via IFN-γ pathway activation. Conclusions and implications L3EMP contributes to LUAD progression through the USP22/SIRT1 signalling axis and represents both a therapeutic target and an immunogenic neoantigen. Targeting L3EMP or its pathway inhibits tumour growth, while the synthetic L3EMP peptide can potentiate immunotherapy. Th