Abstract 7037: Endogenous HiBiT-tagging of PAX3::FOXO1 reveals that CDK inhibitors downregulate the fusion oncogene, and demonstrate synergy effects when combined with vincristine and irinotecan

Abstract BACKGROUND: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancer. PAX3::FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. METHOD: HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the C-terminal end of the endogenous P3F using CRISPR. Western 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 was performed using the Mechanism Interrogation PlatE drug library with known mechanisms of action. Cell viability was measured using CellTiter-Glo. Mouse xenograft models were used to investigate in vivo efficacy. RESULTS: We validated the 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 verified that P3F-HiBiT binds to the same sites as P3F. A screen for compounds that downregulate P3F in both RH4 and SCMC identified 182 drugs. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes, including HDAC inhibitors, BRD4 inhibitors, and CDK inhibitors. Focusing on CDK inhibitors, we found that FP-RMS was most sensitive to CDK7, CDK9 and multi-CDK inhibitors. TG02, a multi-CDK inhibitor with highest inhibition of CDK9 and currently in human trials, downregulated P3F protein. GSEA showed marked suppression of P3F targets after TG02 treatment. Western validated the inhibition of CDK9 with decreased RNA Pol2 Ser2 phosphorylation (Pol2S2). ChIP-seq for RNA Pol2 showed a decrease in transcription pause-release, indicating inhibition of transcription by TG02. Moreover, analysis of genes ranked by decreased Pol2S2 in the gene body showed significant enrichment for P3F targets (p<0.001). TG02 significantly delayed tumor progression without weight loss in a mouse xenograft model of FP-RMS. Also, we found that Vincristine (VCR) and Irinotecan (IRN) are synergistic with TG02 in vitro. Combinations of TG02 with VCR or IRN showed a significant delay in tumor progression compared to TG02 alone in mouse xenograft models. CONCLUSION: By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels. One of the top hits, TG02, showed in vivo efficacy, indicating that FP-RMS is susceptible to multi-CDK inhibition. Decreased occupancy of Pol2S2 in the gene body of P3F targets indicates that the mechanism of TG02 is primarily through transcriptional inhibition of P3F and its targets. This indicates that TG02 may be effective in transcriptionally addicted cancers such as FP-RMS. We also found synergy between TG02 with VCR and IRN showing promise for clinical translation in FP-RMS. Citation Format: Yong Yean Kim, Katrina Jia, Mehal Churiwal, Soumili Dey, Teresa S. Hawley, Silvia Pomella, Raj Chari, David Milewski, Ranuka Sinniah, Young K. Song, Hsien-Chao Chou, Xinyu Wen, Craig J. Thomas, Michele Ceribelli, Jun S. Wei, Robert G. Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3::FOXO1 reveals that CDK inhibitors downregulate the fusion oncogene, and demonstrate synergy effects when combined with vincristine and irinotecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7037.