Targeting E2F8 sensitizes gemcitabine-resistant gallbladder cancer to PARP inhibitors by disrupting RRM2-driven DNA repair

BACKGROUND: Gallbladder cancer (GBC) is an aggressive malignancy with limited therapeutic options, primarily due to the frequent emergence of resistance to gemcitabine-based chemotherapy. Uncovering molecular mechanisms underlying this resistance is essential for developing more effective treatments. METHODS: Gemcitabine-resistant GBC cell lines were generated and subjected to transcriptomic sequencing to identify resistance-associated genes. A genome-wide CRISPR-Cas9 knockout screen was used to pinpoint key genetic regulators. Functional validation was performed through gene knockdown and overexpression, cell viability and apoptosis assays, colony formation, and DNA damage analysis. A high-throughput virtual screening (HTVS) approach was applied to identify small-molecule inhibitors targeting the E2F8-DNA interaction. The efficacy of selected compounds was tested in vitro and in xenograft mouse models, and further validated using patient-derived organoids (PDOs) established from primary and recurrent gallbladder cancers. RESULTS: The transcription factor E2F8 was identified as a driver of gemcitabine resistance via upregulation of RRM2, a gene involved in DNA repair. Knockdown of E2F8 enhanced sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors in resistant GBC cells by impairing DNA repair. HTVS yielded HIT-4, a small-molecule inhibitor that binds to E2F8 and disrupts its interaction with DNA, leading to reduced RRM2 expression. HIT-4 significantly increased apoptosis and DNA damage when combined with PARP inhibitors. In vivo and in PDO models, HIT-4 and PARP inhibitor co-treatment markedly suppressed tumor growth, extended survival, and showed minimal toxicity. CONCLUSIONS: This study identifies the E2F8-RRM2 axis as a key regulator of gemcitabine resistance in GBC and establishes E2F8 as a druggable target. The novel compound HIT-4, in combination with PARP inhibitors, represents a promising therapeutic strategy to overcome chemoresistance and warrants further clinical investigation.