Abstract 4175: Identification of novel inhibitors of the PAX3-FOXO1 fusion oncogene in rhabdomyosarcoma
Authors: Kim Yong Yean , Hawley Robert , Gryder Berkley , Pomella Silvia , Kowalczyk Josh , Sinniah Ranu , Song Young , Khan Javed 
Published: September 2020

Abstract
Background: PAX3/7-FOXO1 fusion gene is the major oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), a highly aggressive soft tissue sarcoma of childhood. The chimeric gene results from either t(2;13) or t(1;13) translocation and has been shown to drive FP-RMS carcinogenesis by the activation of superenhancer driven transcription. Thus, this fusion gene represents a unique vulnerability in FP-RMS which can be targeted by small molecules.
MATERIALS AND METHODS: Novel luciferase assays were developed which simultaneously monitor PAX3-FOXO1 super enhancer and general transcription activity. Using this assay, 62,643 compounds were screened for selective inhibitors of PAX3-FOXO1 activity. RNA-seq was performed on FP-RMS cell lines treated with the top hits and gene set enrichment analysis (GSEA) was performed to assess the molecular effects of the inhibitors. CRISPR-Cas9 lethal screen was performed with and without the top hit molecule to identify synthetic lethal combination to help elucidate the mechanism of action. Western analysis was performed for MYOG, PARP, and PAX3-FOXO1. In vitro direct enzyme inhibition analysis was performed.
RESULTS: PAX3-FOXO1 selective cell-based assay identified 63 compounds that inhibited PAX3-FOXO1 activity without general inhibition of transcription or cell death at 24 hours. Compound PFI-63 was identified as the top hit. RNA-seq and genome wide synthetic lethality studies indicated the compound inhibits histone demethylases. Also, RNA-seq showed activation of apoptosis and myogenic differentiation pathways while PAX3-FOXO1 gene sets were repressed. Activation of apoptosis and myogenic differentiation was validated by Western analysis for PARP cleavage and increase expression of MYOG respectively. CRISPR-Cas9 screen validated the targeting of PAX3-FOXO1 gene sets by PFI-63. In vitro enzymatic inhibition assay confirmed activity of PFI-63 against KDM's with highest specificity to KDM3B. Western analysis for methylation status of histone 3 lysines at positions K27, K4, and K9 showed increase after treatment with PFI-63. However, due to poor solubility of PFI-63, in vivo validation was lacking. Thus, we performed a similarity search of PFI-63 and screened additional compounds leading to the discovery of PFI-90. PFI-90 showed a superior inhibition of KDM family of proteins with highest inhibition of KDM3B via in vitro enzymatic assay. PFI-90 had significantly improved solubility allowing for in vivo administration of drug.
CONCLUSION: We identified two KDM3B selective inhibitors which has activity against FP-RMS in vitro. PFI-63 and PFI-90 disrupts the PAX3-FOXO1 downstream effects and elicits apoptosis and differentiation. Pre-clinical validation by in-vivo experiments is planned. Thus we describe here a novel inhibitor of KDM3B that results in epigenetic inhibition of PAX3-FOXO1 activity representing a potential new therapy for FP-RMS.
Citation Format: Yong Yean Kim, Robert Hawley, Berkley Gryder, Silvia Pomella, Josh Kowalczyk, Ranu Sinniah, Young Song, Javed Khan. Identification of novel inhibitors of the PAX3-FOXO1 fusion oncogene in rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4175.