DNMT3B Knockdown Enhances PARP Inhibitor Sensitivity in Biliary Tract Cancer Cells via Opioid Growth Factor Receptor-Mediated Homologous Recombination Impairment

Background: Biliary tract Cancer (BTC) is an aggressive malignancy with poor prognosis and limited therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated efficacy in tumors with homologous recombination repair (HRR) deficiency. However, actionable BRCA1/2 mutations are rare in BTC. Epigenetic modulation via DNA Methyltransferase (DNMT) inhibition is a proposed strategy for inducing an HR-deficient ("BRCAness") phenotype and thereby enhancing therapeutic response to PARP inhibitors. This study aimed to determine whether the DNMT inhibitor azacitidine (AZA) enhances the antitumor effects of the PARP Inhibitor niraparib (NIR) and to identify molecular mechanisms underlying this interaction. Methods: Two BTC cell lines, TFK-1 and RBE, were treated with AZA and/or NIR or subjected to siRNA-mediated DNMT1, DNMT3A, or DNMT3B knockdown. Functional analyses included homologous recombination (HR) assays, flow cytometric evaluation of cell-cycle distribution and Apoptosis, proliferation and survival assays, and IC₅₀ determination. Whole-transcriptome RNA Sequencing was performed to identify differentially expressed genes after AZA treatment or DNMT3B knockdown, followed by validation via qPCR and Western blotting. To explore epigenetic regulation, whole-genome bisulfite Sequencing was performed on TFK-1 cells following DNMT3B knockdown. Results: AZA treatment decreased HR frequency in a dose-dependent manner and enhanced the sensitivity of BTC cells to NIR, as evidenced by increased Apoptosis, suppressed proliferation, and reduced IC₅₀ values. DNMT3B knockdown recapitulated these effects, establishing a causal relationship between DNMT3B suppression and disrupted HR repair. RNA Sequencing identified opioid growth factor receptor (OGFR) as a commonly upregulated gene after DNMT3B knockdown. Functional validation showed that OGFR overexpression reduced HR activity, increased Apoptosis, and enhanced NIR sensitivity. Contrarily, OGFR knockdown conferred relative resistance. Whole-genome bisulfite Sequencing showed no significant CpG methylation changes at the OGFR promoter region, indicating that OGFR induction is mediated through DNMT3B-dependent transcriptional regulation rather than direct promoter demethylation. Conclusions: DNMT3B inhibition sensitizes BTC cells to PARP inhibitors by disrupting HR repair. OGFR was identified as a novel regulator of HR and PARP Inhibitor sensitivity, controlled via noncanonical DNMT3B-dependent transcriptional mechanisms that operate independently of CpG methylation. These findings provide new mechanistic insights into the epigenetic control of DNA repair and support the rationale for combining DNMT and PARP inhibitors as a promising therapeutic strategy for BTC beyond genetically HR-deficient cases.

DNMT3B; OGFR; PARP inhibitor; azacitidine; biliary tract cancer; epigenetic modulation; homologous recombination; synthetic lethality.