What question did this study set out to answer?

To discover effective small molecule probes that can target the disordered oncoprotein PAX3::FOXO1 associated with fusion-positive ARMS.

April 5, 2026

Abstract 2418: Non-covalent small molecule probe discovery of large intrinsically disordered protein

Key Points

To discover effective small molecule probes that can target the disordered oncoprotein PAX3::FOXO1 associated with fusion-positive ARMS.
Screened a library of small molecules using Small-Molecule Microarrays against PAX3::FOXO1.
Conducted thermal shift assays to assess target engagement of identified compounds.
Developed lentiviral transduced ARMS cell lines expressing HiBiT-tagged PAX3::FOXO1 for validation.
Explored targeted degradation strategies with PROTACs.
Expanded screening libraries to encompass up to 300,000 compounds.
Identified a small molecule that shows selective engagement in thermal shift assay but lacks cellular activity.
Developed scalable protein purification methods for stable recombinant PAX3::FOXO1.
Validated hits using plate-based reporter assays and thermal shift assays.

Abstract

Abstract Fusion-positive alveolar rhabdomyosarcoma (ARMS), is a rare pediatric cancer often characterized by the pathognomonic fusion protein PAX3::FOXO1. Similar to other highly disordered transcription factors, PAX3::FOXO1 lacks typical small-molecule binding pockets thus making drug development difficult by conventional structural oriented design or traditional screening. Currently, there is no direct therapeutic approach to treat children with fusion-positive ARMS and patients are treated with ineffective traditional chemotherapeutic agents. Previously, we screened a library of small molecules against PAX3::FOXO1 via Small-Molecule Microarrays (SMMs) and identified a compound that shows selective target engagement in thermal shift assay but no apparent cellular activity. Furthermore, we are exploring targeted degradation with PROTACs due to the slow turn-over rate of this target. We have identified a scalable protein purification strategy to achieve stable, functional recombinant PAX3::FOXO1 and we are in the process of expanding the SMMs libraries up to 300,000 compounds. To validate hits from those high throughput screenings, we developed lentiviral transduced ARMS cell lines expressing HiBiT-tagged PAX3::FOXO1, enabling plate-based reporter assays as well as thermal shift assays. Small molecules are highly advantageous in their administration, delivery, and patient accessibility. We hope to address some of the unmet needs in the space of small molecule drug discovery against intrinsically disordered oncoprotein and transcription factors. Citation Format: Yichen Xiang, Marc Ramos Sala, Jose I. Vergara Panzone, Nancy Jiang, Diya Rana, Eliot Ebert, Angela N. Koehler. Non-covalent small molecule probe discovery of large intrinsically disordered protein 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 2418.

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