Abstract
Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central “driver” roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.
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Acknowledgments
R.G.H. is a National Institutes of Health (NIH) Research Collaborator. This work was initiated during a sabbatical as a Special Volunteer with Javed Khan, Deputy Director, Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD. We are thankful to him for continued support. The authors thank Raj Chari and Christine Evans, Genome Modification Core, Frederick National Laboratory for Cancer Research for designing and providing CRISPR plasmids. The authors are grateful to Silvia Pomella and Young Song for assistance with western blotting and RNA-seq experiments, respectively. The authors also appreciate Craig Thomas and Michele Ceribelli, Division of Preclinical Innovation, Chemistry Technologies, National Center for Advancing Translational Sciences, NIH for their willingness to establish and perform high-throughput drug screens of RH4.P3F-HmC 1A9 and SCMC.P3F-HmC 3C4 cells using the Nano-Glo HiBiT Lytic Detection System.
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Hawley, R.G., Hawley, T.S. (2024). CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting. In: Hawley, T.S., Hawley, R.G. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 2779. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3738-8_12
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