Chordoma Foundation

Abstract Chordoma is a rare bone cancer with a high rate of disease recurrence and no approved systemic therapies. Tumor profiling and functional genomics studies suggest a significant number of chordomas are characterized by molecular alterations associated with increased replication stress, and a dependence on replication stress response pathways. We confirm here in preclinical models that chordomas exhibit a replication stress phenotype, which creates an actionable therapeutic vulnerability exploitable by induction of replication stress and/or inhibition of the ATR-dependent replication stress response. A panel of chordoma cell lines was treated with gemcitabine or the ATR inhibitor (ATRi) elimusertib (BAY 1895344). In a subset of cell lines, gemcitabine or elimusertib demonstrated low-nanomolar EC50 values. Upon treatment with the ATRi 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 inhibition, suggesting possible fork stalling or collapse. Alkaline and neutral comet assays were performed to determine the presence of genome-wide ssDNA and/or dsDNA breaks, respectively, with orthogonal evaluation of H2A.X activation during S-phase to assess DNA strand integrity after ATR and DNA synthesis inhibition. 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. PDX models sensitive to ATR inhibition were also highly sensitive to gemcitabine, further suggesting gemcitabine induces an ATR-dependent replication stress response in chordoma models. Indeed, combining gemcitabine with ATRi revealed exceptional synergy in chordoma cell lines. Collectively, our data indicate that a subset of chordomas are characterized by intrinsic replication stress that can be therapeutically exploited through the synergistic activity of gemcitabine and ATRi. Citation Format: Nindo Punturi, Wendy Leung, Joan Levy, Lee Zou, Gregory M. Cote, Dan M. Freed. Assessing replication stress as an actionable therapeutic opportunity in chordoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B021.

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.

Abstract Background: Chordoma is a rare cancer (0.08 per 100k/yr) that originates from the notochord and develops in the skull and spine. Treatment options for chordoma include resection and local radiation therapy; however, recurrence rates following treatment are high and the majority of patients ultimately succumb to the disease. Currently there are no approved chemotherapy options for chordoma, and effective treatment options for patients with recurrent or advanced disease are limited. Recent molecular analyses of chordoma have revealed multiple tractable therapeutic targets, providing rationale for new systemic therapies. However, lack of relevant, validated chordoma models has limited preclinical evaluation. To address this need, the Chordoma Foundation and START have collaborated to establish, bank and characterize chordoma tumor models derived from patient samples and established cell lines. Methods: Chordoma patients undergoing resection or biopsy procedures are identified and consented by the Chordoma Foundation under an IRB-approved protocol; tissue is shipped to START and implanted into immune-deficient mice for patient-derived xenograft (PDX) model establishment and banking. Similarly, models from immortalized chordoma cell lines have been established and banked, along with established PDX chordoma models from START or collaborators. Banked PDX models are anonymously linked to available patient clinical information. All chordoma models are subjected to immunohistochemistry (IHC) to confirm histology and presence of brachyury, a protein highly expressed in chordoma tumors. Following validation, models are used at START for drug sensitivity studies and available to investigators for characterization studies. Results: To date, clinical samples from five chordoma patients have been implanted and two PDX models designated CF322 and CF345 have been established. In addition, xenograft models from the UCH-1 and UCH-2 chordoma cell lines have been established at START. PDX models previously developed at START (ST087) and UCSF (SF8894) have also been validated and banked. Drug sensitivity studies have been initiated these two and the U-CH1 models evaluating agents selected and prioritized through a peer review process established by the Chordoma Foundation. Nominations for additional test agents from academic and industry collaborators are reviewed on a rolling basis and evaluation of selected therapies is funded by the Chordoma Foundation. Conclusion: We have established a collaboration to create, bank and characterize a panel of preclinical chordoma tumor models originating from patient samples and established cell lines. To date we have generated three chordoma models available for drug sensitivity studies. This panel of models is intended to serve as a shared resource to the chordoma research community to enable more rapid preclinical evaluation of therapeutic hypotheses. Citation Format: Michael J. Wick, Melissa Rundle, Lizette Gamez, Armando Diaz, Josh Sommer, Patricia Cogswell, Byron Hann, Joanna Phillips, Kyriakos P. Papadopoulos. Establishment and characterization of a panel of cell-based and patient-derived chordoma tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 627.

It is not uncommon for potentially life-saving research data to be published years after being generated. But the setback to progress caused by the delay in releasing data is troublesome for people who selflessly participate in trials and desperately await new therapies. Scientists need to feel greater urgency to share their findings quickly, and they need additional avenues to facilitate this process.