Abstract B034: A chemoproteomic-enabled phenotypic library screen identifies KIF23 as a novel target in Ewing sarcoma

Abstract Ewing sarcoma (ES) is a bone and soft tissue malignancy that presents clinical challenges due to the lack of targeted treatments. The current standard of care involves a combination of surgery, radiation, and chemotherapeutic agents. Despite these interventions, overall survival rates for patients with advanced or relapsed ES remain dismal, highlighting the critical need for novel, targeted therapeutics. Given the reliance of ES on the aberrant transcription factor fusion protein EWS-FLI1, we hypothesize that the downstream signaling pathways present an exploitable opportunity for targeted therapeutic development. To identify core factors driving the oncogenic program in ES, we employed a fragment-based phenotypic screening approach. A diverse library of 640 fully functionalized fragment-like probes were screened using CellTiter-Glo to assess cell viability. The library consisted of a biocore fragment coupled to a short linker possessing a ‘clickable’ alkyne and photoactivatable diazirine group for target protein identification by chemical proteomics. We identified several probes that inhibited cell proliferation in ES cells compared to other cancer cell types. Network similarity analysis revealed distinct scaffolds, predominantly benzofurans and benzimidazole/indazoles. We identified novel ES targets using the identified hits by MS-based chemoproteomics using UV-mediated photo crosslinking of targets, followed by copper-catalyzed azide-alkyne cycloaddition ‘click’ chemistry, enabling biotin-mediated enrichment. We identified kinesin family member 23 (KIF23) as a target of interest. Using genetic studies, we validated that KIF23 knockdown has significant and selective effects on ES viability when compared to other cancer types. The initial round of structure-activity relationship studies focused on diazirine substitution strategies, aiming to refine pharmacophores and optimize compounds for cellular applications. Further studies will focus on understanding the role of KIF23 in ES and its relationship with the driver fusion protein EWS-FLI1, as well as hit-to-lead optimization of the chemistry of the identified fragments. Citation Format: Hannah L. Walker-Mimms, Nada Elkholy, Yi Liao, Micheal Lamptey, Xueli Li, Nicole Londono, Uwe Rix, Andrii Monastyrskyi, Derek Duckett. A chemoproteomic-enabled phenotypic library screen identifies KIF23 as a novel target in Ewing sarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B034.