CUL3-mediated NRF2 dysregulation in TP53-driven non-small cell lung cancer: neddylation-associated structural perturbations

CUL3 encodes Cullin-3, the scaffold of Cullin-RING Ligase 3 (CRL3), which mediates proteasomal degradation of NRF2 through KEAP1-dependent ubiquitination. While canonical NRF2 alterations such as KEAP1 or NFE2L2 mutations are well characterised in non-small cell lung cancer (NSCLC), the role of CUL3 mutations remains poorly defined. We hypothesised that CUL3 missense variants are conditionally enriched in TP53-altered NSCLC, with the EGFR-oncogenic subset as a defined disease background rather than a synergistic requirement. A multi-cohort NSCLC dataset (n = 47,862; 10,228 with oncogenic EGFR mutations and confirmed CUL3 coverage) was interrogated using two-layer Fisher’s exact testing and logistic regression with an EGFR × TP53 interaction term, and validated transcriptomically in TCGA LUAD/LUSC. CUL3 mutations showed conditional enrichment in the TP53-mutant EGFR background (OR = 2.92, p = 4.68 × 10 −29 ); the non-significant EGFR × TP53 interaction (OR = 0.86, p = 0.547) confirmed that enrichment is driven by TP53 loss rather than EGFR synergy. CUL3-mutant tumours showed elevated NRF2 activation across three independent signatures, with increased proliferative signalling and altered drug metabolism. All-atom molecular dynamics simulations of wild-type, L321F, G402E and M711I CUL3:KEAP1 complexes in inactive and neddylation-active states revealed variant-specific conformational behaviour. L321F drove the neddylated complex into a conformationally restricted state with reduced backbone flexibility, decreased radius of gyration, and a shifted free energy minimum, whereas G402E showed progressive destabilisation and M711I remained comparable to wild-type. Free energy landscape analysis confirmed a shifted conformational energy minimum in L321F + NEDD8 relative to wild-type, indicating impaired catalytic flexibility despite intact complex formation. These findings define CUL3 as a TP53-associated modifier of NRF2 regulation within EGFR-driven NSCLC, operating through allosteric rigidification of the neddylation-active CRL3 complex.