Elucidation of breakage-fusion-bridge (BFB)-mediated EGFR amplification in lenvatinib-resistant hepatocellular carcinoma

Lenvatinib, a multi-targeted tyrosine kinase inhibitor, is widely used as a first-line therapy for patients with immunotherapy-ineligible hepatocellular carcinoma (HCC). However, its clinical benefit is often compromised by the development of resistance. Understanding the genomic alterations driving resistance is critical to improving therapeutic outcomes in HCC. In this study, a lenvatinib-resistant HCC cell line, Huh7, was established by exposing parental sensitive cells to increasing concentrations of lenvatinib. To elucidate the mechanisms underlying drug resistance, short- and long-read whole genome sequencing (WGS), whole transcriptome sequencing (WTS), and western blot were performed. Comparative analysis of genomic alterations between parental and resistant cells revealed marked differences in copy number (CN) profiles. In resistant cells , EGFR showed the most prominent CN gain, increasing from 6 to 21 copies. Detailed breakpoint analysis utilizing LUMPY identified fold-back inversions that directly corresponded with genomic locations exhibiting significant CN alterations, indicative of the hallmark features of breakage-fusion-bridge (BFB) cycles. These findings were confirmed by Sniffles-based long-read SV calling. Transcriptomic and protein-level analyses using WTS and Western blotting, respectively, showed increased EGFR expression. Gene set enrichment analysis showed significant enrichment of ERBB and MAPK pathways. Mechanistically, activation of the EGFR-PAK2-ERK5 axis was observed, suggesting that lenvatinib-induced blockade of VEGFR/FGFR promotes a bypass survival signal through EGFR downstream pathways. Combined treatment with lenvatinib and erlotinib, an EGFR inhibitor, reduced viability and restored sensitivity. Clinically, lenvatinib-treated HCC patients with high EGFR immunohistochemistry scores exhibited shorter progression-free survival, and public HCC cell line datasets consistently showed poor responses in EGFR -amplified cells. In conclusion, our findings suggest BFB-driven EGFR amplification as a key structural and functional driver of lenvatinib resistance in HCC. Lenvatinib combined with an EGFR inhibitor may offer a treatment option for patients with HCC exhibiting elevated EGFR expression.