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Abstract B56: A CRISPR/Cas9 domain screen identifies a small motif in the PAX3-FOXO1 transactivation domain relevant for tumor maintenance in alveolar rhabdomyosarcoma

Fusion transcription factors are the main drivers of tumorigenesis in many pediatric leukemias and sarcomas. One example is PAX3-FOXO1 (P3F), the pathognomonic marker of alveolar rhabdomyosarcoma (aRMS). Since these tumors are often also characterized by a quiet mutational landscape and a paucity of druggable oncogenes, the fusion proteins often remain as the only relevant drug target. However, transcription factors are challenging targets for small-molecule inhibitors, since they lack an enzymatic activity and, apart from the DNA binding domains, are usually intrinsically disordered. Identification of druggable structures in such proteins is therefore of great clinical interest. Our goal of this study was to identify new elements important for P3F activity. Towards this aim, we made use of the CRISPR/Cas9 technology and performed a saturated screen for identification of important functional domains in P3F in the endogenous context. This revealed that both DNA binding domains are indispensable, as expected for a transcription factor. Importantly, however, the screen also identified a 40-amino-acid-long domain at the C-terminus of the FOXO1 part to be strictly required for the function of the protein. Using a series of mutations in this domain in reporter assays, we identified a single cysteine whose mutation leads to more than 50% reduction in transcriptional activity. In addition, cells efficiently differentiated and stopped proliferating when this Cys was mutated in aRMS cells. These results suggest that functional interference with this domain could be a promising approach for aRMS therapy. Citation Format: Marco Wachtel, Katharina Benischke, Beat W. Schafer. A CRISPR/Cas9 domain screen identifies a small motif in the PAX3-FOXO1 transactivation domain relevant for tumor maintenance in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B56.