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

## TL;DR

This study finds that targeting E2F8 can make gallbladder cancer cells resistant to gemcitabine more sensitive to PARP inhibitors by disrupting DNA repair.

## Contribution

The study identifies E2F8 as a novel druggable target and introduces HIT-4, a new compound that enhances PARP inhibitor efficacy in resistant gallbladder cancer.

## Key findings

- E2F8 drives gemcitabine resistance by upregulating RRM2, a DNA repair gene.
- Knocking down E2F8 increases sensitivity to PARP inhibitors by impairing DNA repair.
- HIT-4, a small-molecule E2F8 inhibitor, combined with PARP inhibitors, significantly reduces tumor growth in models.

## Abstract

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.

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.

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.

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.

The online version contains supplementary material available at 10.1186/s13046-025-03586-2.