Colorectal cancer (CRC) remains a major global health challenge, with fewer than 20% of patients with metastatic disease surviving beyond five years. Anti-EGFR monoclonal antibodies such as cetuximab or panitumumab, combined with fluoropyrimidine-based doublet chemotherapy are standard of care for patients with RAS/BRAF wild-type tumors, providing significant clinical benefit. Nevertheless, their efficacy is often limited by acquired resistance, which drives disease progression. Although several mechanisms of resistance have been described, the molecular pathways underlying therapeutic escape remain incompletely understood.

Two panitumumab-resistant CRC models were generated by exposing the highly sensitive LIM1215 cell line to fixed or escalating drug concentrations. Resistant cells were characterized using functional assays and integrated multi-omics approaches, including genomics, proteomics, phosphoproteomics and metabolomics.

Resistant cells exhibited increased proliferation, migration, 3D growth and metabolic activity compared with parental cells. Genomic profiling revealed an activating KRAS exon 4 (A146) mutation in both models and an additional NRAS exon 3 (Q61R) mutation in one. Metabolomic analyses revealed extensive metabolic rewiring characterized by enhanced glycolysis, altered amino acid metabolism and increased bioenergetic capacity. Proteomic and phosphoproteomic profiling demonstrated sustained activation of ErbB signaling, PI3K/AKT/mTOR, and STAT3 pathways, along with remodeling of cytoskeletal, extracellular matrix, DNA repair and growth factor-related pathways, despite EGFR blockade. Upstream regulator analysis suggested that resistance is maintained by coordinated transcriptional, metabolic, signaling and chromatin-associated networks beyond individual pathways. These findings provide a rationale for targeting adaptive epigenetic plasticity and supports the evaluation of histone deacetylase inhibitors (HDACi) in combination with anti-EGFR therapy. Notably, we observed that treatment with the HDACi valproic acid partially restored panitumumab sensitivity, impaired clonogenic and spheroid growth, and reduced glycolytic ATP production in resistant cells.

Our multi-omics framework identifies epigenetic plasticity and metabolic adaptation as key vulnerabilities in panitumumab-resistant CRC, supporting the therapeutic potential of HDAC inhibition. These findings provide preclinical evidence for combining HDACi with EGFR-targeted therapies to overcome secondary resistance in CRC and guide future translational investigations.

This work is supported by European Union–NextGenerationEU initiative under the Italian Ministry of Health (PNRR M6/C2_CALL 2023, Project PNRR-MCnT2-2023-12377998).