ABTB1 modulates EGFR signaling and confers icotinib sensitivity in lung adenocarcinoma

Icotinib is a first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) widely used in the treatment of EGFR-mutant lung adenocarcinoma (LUAD). However, heterogeneous responses and the development of resistance limit durable clinical benefit. The molecular regulators that modulate EGFR signaling output and influence cellular sensitivity to icotinib remain incompletely defined. An integrative discovery framework was implemented by combining genome-wide CRISPR–Cas9 functional screening under icotinib selection pressure with RNA sequencing analyses performed under basal and icotinib-treated conditions. Candidate regulators were prioritized through multi-layer integration of functional dependency, transcriptional response, and disease-context expression data. Clinical associations were evaluated using public LUAD datasets. Functional validation was performed using cell proliferation assays, dose–response analyses, and biochemical approaches. Protein interaction and signaling mechanisms were examined using immunoprecipitation–mass spectrometry, co-immunoprecipitation, immunofluorescence, and molecular docking analyses. Integrated functional and transcriptomic analyses identified ABTB1 as a candidate regulator associated with icotinib response. ABTB1 expression was reduced in LUAD and correlated with aggressive tumor features and unfavorable overall survival. Functionally, ABTB1 overexpression enhanced cellular sensitivity to icotinib, as evidenced by suppressed proliferation, leftward shifts of dose–response curves, and reduced IC50 values. Proteomic profiling identified EGFR as a prominent ABTB1-associated protein, which was further validated by biochemical and spatial co-localization analyses. Mechanistically, ABTB1 overexpression attenuated EGFR phosphorylation and downstream STAT3 and NF-κB signaling, accompanied by coordinated transcriptional remodeling of gene programs related to stress response, inflammatory signaling, and cell fate regulation. This study identifies ABTB1 as a non-mutational regulator of EGFR signaling that modulates cellular sensitivity to icotinib in LUAD. By integrating functional screening, transcriptomic network analysis, and mechanistic validation, our findings reveal a receptor-proximal control layer shaping icotinib response and highlight ABTB1 as a potential biomarker and modulatory factor in icotinib-treated LUAD.