Abstract 4321: Selective targeting of TBXT with DARPins identifies regulatory networks and therapeutic vulnerabilities in chordoma

Abstract Aberrant expression of the embryonal transcription factor TBXT (also known as brachyury) drives chordoma, a rare spinal neoplasm with no effective drug therapies. The gene network regulated by TBXT is poorly understood, and strategies to disrupt its abnormal activity for therapeutic purposes are lacking. We developed TBXT-targeted designed ankyrin repeat proteins (T-DARPins) that selectively bind TBXT and inhibit its binding to DNA. In chordoma cells, T-DARPins reduced cell cycle progression, spheroid formation, and tumor growth in mice. Combining T-DARPin-mediated TBXT inhibition with transcriptomic and proteomic analyses, we determined the TBXT regulome in UM-Chor1 chordoma cells, which comprises in particular networks involved in cell cycle regulation, DNA replication and repair, embryonal cell identity, metabolic processes, and interferon response. The analysis of selected TBXT regulome components provided new insights into chordoma biology, such as the strong upregulation of IGFBP3 upon TBXT inhibition to fine-tune part of TBXT’s downstream effectors. Finally, we assigned each TBXT regulome member a druggability status to create a resource for future translational studies and found high interferon response signaling in chordoma cell lines and patient tumors, which was promoted by TBXT and associated with strong sensitivity to clinically approved JAK2 inhibitors. These findings demonstrate the potential of DARPins to interrogate the function of nuclear proteins to understand the regulatory networks of cancers driven by aberrant transcription factor activity, including novel entry points for targeted therapies that warrant testing in patients. Citation Format: Charles S. Umbaugh, Marie Groth, Cihan Erkut, Kwang-Seok Lee, Joana Marinho, Florian Iser, Jonas N. Kapp, Petra Schroeter, Simay Dolaner, Asli Kayserili, Julia Hartmann, Philipp Walch, Thomas F. Barth, Kevin Mellert, Birgit Dreier, Jonas V. Schäfer, Andreas Plückthun, Stefan Fröhling, Claudia Scholl. Selective targeting of TBXT with DARPins identifies regulatory networks and therapeutic vulnerabilities in chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4321.