Abstract 1089: Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine
Authors: Yong Yean Kim, Katrina Jia, Mehal Churiwal, Teresa Hawley, Silvia Pomella, Christine Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young Song, Hsien-Chao Chou, Xinyu Wen, Craig Thomas, Michele Ceribelli, Jun Wei, Robert Hawley, Javed Khan
Journal: Cancer Research (2024)

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens. Method: HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR in FP-RMS cell lines RH4 and SCMC. Western blot was used for HiBiT tag validation. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT). High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, a 2,480 drug library with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. Mouse xenograft models of FP-RMS were used to investigate in vivo efficacy. Results: We validated HiBiT tagging of P3F by Western. Both P3F-HiBiT and unmodified P3F activated the same gene sets in fibroblasts by RNA-seq Gene Set Enrichment Analysis (GSEA). ChIP-seq using HiBiT antibody for P3F-HiBiT matched the genomic locations from ChIP-seq with P3F antibody in RH4 and SCMC. Using a cutoff value of &amp;gt; 90 (Area Under the Curve (AUC) of CellTiter-Glo minus AUC of Nano-Glo), in both RH4 and SCMC, we identified 182 compounds which downregulate P3F before cell death. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F including HDAC inhibitors (3), BRD4 inhibitors (3), and CDK inhibitors (8). FP-RMS was sensitive to CDK1/2, CDK4/6, CDK9, and multi-CDK inhibitors. A multi-CDK inhibitor TG02, currently in human trials, downregulated P3F and RNA-seq GSEA showed marked suppression of P3F targets. TG02 also significantly delayed tumor progression in mouse xenograft model of FP-RMS without weight loss. Interestingly, the commonly used chemotherapeutic Vincristine (VCR) also downregulated P3F in vitro. TG02 with VCR showed synergy by Loewe analysis. In vivo, testing showed significant delay in tumor progression by the combination compared to TG02 or VCR alone. Of note, tumor RNA-seq GSEA following treatment with TG02, or VCR alone, and in combination significantly downregulated P3F targets. Conclusion And Future Directions: By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels of which TG02 was a top hit. TG02 showed in vivo efficacy indicating that FP-RMS is susceptible to CDK inhibition. We also found synergy between TG02 and VCR, resulting in a significant delay in FP-RMS tumor progression compared to single agents. Interestingly, we found that VCR alone can downregulate P3F and its targets. Combination therapy of TG02 with VCR shows promise for clinical translation in FP-RMS. Citation Format: Yong Yean Kim, Katrina Jia, Mehal Churiwal, Teresa Hawley, Silvia Pomella, Christine Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young Song, Hsien-Chao Chou, Xinyu Wen, Craig Thomas, Michele Ceribelli, Jun Wei, Robert Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1089.