Abstract
Gemcitabine-based chemotherapy remains the standard first-line treatment for cholangiocarcinoma (CCA), but acquired resistance presents a significant clinical challenge. Synthetic lethality approaches targeting double-strand break repair (DSBR) pathways offer promising therapeutic opportunities. Ataxia-telangiectasia mutated (ATM) kinase, a central regulator of homologous recombination (HR) and non-homologous end joining (NHEJ), is critical for maintaining genomic integrity following DNA damage. Here, we demonstrate that combining the ATM inhibitor AZD0156 with DNA-damaging agents (cisplatin or photon irradiation) significantly enhances cytotoxicity in gemcitabine-resistant intrahepatic CCA sublines (GR-iCCAs) while sparing gemcitabine-sensitive parental cells. This selective sensitization manifests in impaired colony formation, increased apoptosis, and persistent γ-H2AX nuclear accumulation. The magnitude of AZD0156 sensitization in GR cells substantially exceeds additive expectations, strongly suggesting synergistic interaction. Genetic ATM depletion in GR-iCCAs under genotoxic stress recapitulated these effects, confirming on-target specificity. Mechanistically, GR-iCCAs exhibit significantly reduced DNA ligase I (LIG1) expression, a critical component of the alternative NHEJ (alt-NHEJ) repair pathway, particularly under DNA damage conditions. Genetic restoration of LIG1 expression reversed AZD0156 sensitivity, establishing LIG1 deficiency as a key determinant modulating DNA repair pathway dependency. In xenograft models, AZD0156 combined with cisplatin substantially suppressed tumor growth compared to monotherapy, with acceptable tolerability profiles. These findings identify ATM inhibition as a promising strategy to overcome gemcitabine resistance in CCA, particularly in tumors with compromised alt-NHEJ repair capacity, providing a mechanistic rationale for clinical development of this combination therapy.