Assessing replication stress as an actionable therapeutic opportunity in chordoma

Abstract Chordoma is a rare bone cancer with a high rate of disease recurrence and no approved systemic therapies. Tumor profiling studies indicate a significant number of chordomas are characterized by alterations in SWI/SNF chromatin remodeling and DNA damage repair genes, which have been associated with replication stress in other cancer types. We demonstrate here that preclinical chordoma models exhibit a replication stress phenotype and dependence on ATR activity. To assess the effect of ATR inhibition on cell viability, a panel of chordoma cell lines were treated with the ATR inhibitor elimusertib (BAY 1895344). In a subset of cell lines, elimusertib inhibited viability with low-nanomolar IC50 values. Upon treatment with the ATR inhibitor ceralasertib (AZD6738), DNA fiber assays revealed a mild decrease in replication fork speed and a significant increase in fork asymmetry in cells sensitive to ATR inhibitor, suggesting possible fork stalling or collapse in response to ATR inhibition. To assess genomic instability as a consequence of ATR inactivation and fork stalling or collapse, alkaline and neutral comet assays were performed to determine the presence of genome-wide ssDNA and/or dsDNA breaks, respectively. In a panel of chordoma PDX models, striking differential sensitivity to ATR inhibition was observed. In sensitive models, elimusertib (50 mg/kg, po, bidx3) promoted 85-90% tumor growth inhibition, in contrast to resistant models, where elimusertib treatment had no effect on tumor growth. Moreover, PDX models sensitive to ATR inhibition were sensitive to other therapies that exacerbate replication stress; for example, gemcitabine promoted multiple complete responses in a pediatric chordoma PDX model. Coupled with preclinical efficacy data, genomic and transcriptomic sequencing of differentially-sensitive chordoma models provided insight into the molecular factors associated with the replication stress phenotype in chordoma - illuminating potential biomarkers for patient selection. Finally, we explore various combination therapies and show that combining elimusertib with cisplatin or gemcitabine synergistically inhibits the viability of chordoma cell lines. Collectively, our data indicate that a subset of chordomas are characterized by intrinsic replication stress that can be therapeutically exploited by inhibiting ATR. Citation Format: Nindo Punturi, Lee Dolat, Wendy Leung, Joan Levy, Lee Zou, Daniel M. Freed, Gregory M. Cote. Assessing replication stress as an actionable therapeutic opportunity in chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6195.