CD24 is a promising immunotherapeutic target for enhancing efficacy of third‐generation EGFR‐TKIs on EGFR‐mutated lung cancer

Abstract Background Third‐generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) show initial efficacy in EGFR‐mutated lung cancer, but residual disease persists. This study aimed to investigate cluster of differentiation 24 (CD24) as a translational immunotherapeutic target for enhancing third‐generation EGFR‐TKI efficacy. Methods We conducted RNA‐sequencing (RNA‐seq) on drug‐responsive, drug‐tolerant persister, and drug‐resistant cells to identify therapeutic targets to pair with EGFR‐TKIs. For validation, we integrated single‐cell RNA‐seq data from 29 lung cancer specimens and used single‐nucleus RNA‐seq and immunohistochemistry on clinical residual tumor samples following TKI therapy (TKI‐residual). With CRISPR/Cas9, we studied the effect of CD24 on proliferation and phagocytic clearance during EGFR‐TKI treatment. We tested CD24 knockout or ATG‐031 (a first‐in‐class CD24 antibody) with EGFR‐TKIs in vitro, xenografts, and spontaneous lung cancer models. To explore mechanisms, we used DNA affinity precipitation, chromatin immunoprecipitation sequencing, and luciferase assays to identify transcription factors regulating CD24. Co‐immunoprecipitation combined with mass spectrometry and phosphoproteomics were used to study YIN‐YANG‐1 (YY1) S247 phosphorylation's expression and function, while kinase inhibitors assessed upstream phosphorylation of YY1 S247 and its regulation of CD24. Results CD24 expression rose in drug‐responsive, ‐resistant, and ‐tolerant lung cancer cells and post‐EGFR‐TKI treatment clinical specimens. This elevation promoted cell proliferation and shielded tumor cells from macrophage‐mediated phagocytosis. Genetic depletion of CD24 or treatment with ATG‐031 significantly enhanced phagocytosis and tumor eradication in vitro, in xenografts, and in mice harboring EGFRL858R·T790M‐driven spontaneous lung tumors. Furthermore, we revealed that YY1 S247 phosphorylation was responsible for the upregulation of CD24 upon EGFR‐TKI treatment, facilitating YY1 dimerization and the formation of promoter‐enhancer loops that regulate CD24 expression. Conclusions CD24 is a promising target in EGFR‐mutated lung cancers, potentially enhancing efficacy of third‐generation EGFR‐TKIs.