Abstract 252: Targeting replication stress promotes immunogenic cell death in chordoma.

Abstract Chordoma is a rare bone cancer with a high rate of recurrence and no approved systemic therapies. Target discovery efforts have uncovered complex genomic rearrangements, alterations in DNA damage response (DDR) and chromatin-remodeling genes, and a reliance on DDR pathways that stabilize stalled replication forks. Additionally, a subset of chordomas is highly infiltrated by multicellular immune aggregates, but the underlying mechanisms driving an antitumor immune response remain unclear. We hypothesized that replication stress presents an actionable therapeutic vulnerability in chordoma and that targeting this pathway could induce lethal DNA damage and immunogenic cell death. To assess the effects of exacerbating replication stress in chordoma, a panel of 14 cell lines was treated with the DNA synthesis inhibitor gemcitabine or the ATR inhibitor elimusertib (BAY 1895344). Most lines were highly sensitive, with 9 of 14 showing EC50 values below 10 nM (gemcitabine) or 100 nM (elimusertib). Biochemical analysis showed that gemcitabine-induced DNA damage leads to activation of Chk1 in an ATR-dependent manner, with DNA fiber assays revealing decreased replication fork speed, symmetry, and stability following ATR inhibition (ATRi). Sensitivity to ATRi or gemcitabine was mirrored in chordoma PDX models, with ATRi promoting 85–90% tumor growth inhibition in sensitive models and gemcitabine driving tumor regressions. Exceptional synergy was observed in vitro when these therapies were combined, with significant reductions in cell viability, enhanced DNA double-strand breaks, and near-complete S-phase collapse. Moreover, treatment with either drug resulted in the accumulation of double-stranded DNA in the cytoplasm, along with upregulation of type I interferon, immunomodulatory chemokines CXCL10 and CCL5, and cell surface PD-L1. These findings are consistent with a model where ATRi or gemcitabine treatment promotes lethal DNA damage and immunogenic cell death in chordoma, which may be further augmented by PD-1 checkpoint blockade. Citation Format: Nindo Punturi, Arijit Ghosh, Caitlin King, Wendy Leung, Joan B. Levy, Lee Zou, Gregory M. Cote, Dan Freed. Targeting replication stress promotes immunogenic cell death in chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 252.