CRISPR/Cas9 library screening uncovered CCT2 as a critical driver of acquired resistance to EGFR-targeted therapy by stabilizing TMX1 in non-small cell lung cancer

n the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor 27receptor tyrosine kinase inhibitors (EGFR-TKIs), the emergence of acquired 28resistance remains a significant challenge. Elucidating the underlying mechanisms of 29resistance is crucial for developing novel strategies to overcome or delay therapeutic 30escape. To this end, this study aimed to identify key drivers of EGFR-TKIs resistance 31and explore actionable targets for intervention. We investigated resistance 32mechanisms by integrating CRISPR/Cas9-based genome-wide screening with tandem 33mass tag (TMT) proteomic analysis, and virtually screened bioactive small molecule 34libraries to identify compounds capable of restoring EGFR-TKIs sensitivity. The 35multi-omics approach revealed that CCT2 is a critical mediator of resistance to 36third-generation EGFR-TKIs in lung cancer, with higher expression of CCT2 37observed in resistant cells compared to sensitive cells. Mechanistically, CCT2 recruits 38tripartite motif-containing protein 28 (TRIM28) to catalyze SUMO2 modification of 39thioredoxin-related transmembrane protein 1 (TMX1), inhibiting its ubiquitination 40and enhancing protein stability. This post-translational modification (PTM) promotes 41TMX1-dependent reactive oxygen species (ROS) clearance, thereby conferring 42resistance. Importantly, pharmacological inhibition with the compound HY-10127, 43identified through virtual screening, effectively restored EGFR-TKIs sensitivity in 44resistant cell lines and delayed the development of resistance in xenograft models. 45The findings establish the CCT2/TRIM28/TMX1/ROS axis as a novel resistance 46mechanism in EGFR-mutated lung cancer, and targeting this pathway with HY-10127 47represents a promising strategy to overcome resistance to third-generation 48EGFR-TKIs, providing preclinical rationale for clinical translation. These discoveries 49advance our understanding of molecular resistance mechanisms and offer potential 50therapeutic targets for improving lung cancer prognosis.