Circulating epigenetic and epitranscriptomic biomarkers identify EGFR status, predict TKI response and reveal...

Circulating cell-free epigenetic and epitranscriptomic modifications (epimarks) are emerging as minimally invasive biomarkers for cancer detection and therapeutic monitoring. Their clinical utility in non-small cell lung cancer (NSCLC), particularly in the context of EGFR-targeted therapies, remains to be fully established. Ten circulating epimarks encompassing cfRNA methylation, histone post-translational modifications on circulating nucleosomes, and cfDNA methylation were quantified in plasma samples from healthy donors and metastatic NSCLC patients with mutant or wild-type EGFR. Longitudinal analyses were performed in EGFR-mutant patients treated with tyrosine kinase inhibitors (TKIs). Functional relevance was assessed using in vitro and in ovo models of Osimertinib-sensitive and -resistant NSCLC cells combined with targeted epigenetic inhibitors. Distinct epimark signatures accurately discriminated healthy donors from NSCLC patients and distinguished EGFR-mutant from EGFR-wild-type tumors. cfRNA methylation marks (m1A, m6A, m5C, and m7G) showed excellent diagnostic performance, with a composite methyl-cfRNA score robustly distinguishing EGFR-mutant NSCLC. Disease progression under TKI therapy was characterized by increased m1A-cfRNA, H3K23Ac- and H4R3me2-circulating nucleosomes and decreased 5mC-cfDNA, with m1A-cfRNA alone reliably tracking progression. High m6A-cfRNA levels at diagnosis were strongly associated with shorter progression-free survival. Functionally, Osimertinib resistance promoted tumor growth and metastatic dissemination. Combination therapies targeting METTL3, KAT6A, or PRMT5 with Osimertinib reduced tumor burden and metastasis through miRNA-dependent regulation of histone acetylation and methylation. Circulating epimarks represent promosing diagnostic and prognostic biomarkers and provide a mechanistic basis for epigenetic combination therapies in EGFR-driven NSCLC.