Abstract 703: Novel histone lysine demethylase inhibitors disrupt PAX3-FOXO1-driven transcriptional output in fusion-positive rhabdomyosarcoma

Abstract BACKGROUND: The PAX3/7-FOXO1 (P3F) fusion transcription factor is the oncogenic driver in fusion-positive rhabdomyosarcoma (FP-RMS). P3F drives oncogenesis in FP-RMS through transcriptional modulation of downstream target genes. Thus, P3F represents a unique vulnerability in FP-RMS. A screen for inhibitors of P3F action identified novel histone lysine demethylase (KDM) inhibitors characterized in this study. MATERIALS/METHODS: A specific luciferase assay which monitors P3F activity was utilized to screen 62,643 compounds. The top candidate with unknown mechanism of action, PFI-63, and its analog PFI-90, were characterized. Western blotting was used to detect MYOG, PARP, and PAX3-FOXO1. KDM enzyme inhibition assays were conducted. Ligand-observed NMR analysis was used to determine binding of PFI-90 to KDM3B. KDM knockdown using CRISPRi was carried out. ChIP-seq analysis on H3K4me3, H3K9me2, H3K27me3, H3K27ac, and PAX3-FOXO1 was performed. Mouse xenograft models of FP-RMS were used to determine in vivo efficacy. RESULTS: 64 compounds that inhibited P3F activity without general inhibition of transcription or induction of cell death were further characterized. PFI-63 and a more water-soluble analog, PFI-90, were identified. GSEA of RNA-seq showed activation of apoptosis and myogenesis while P3F targets were repressed. Activation of apoptosis and myogenesis were validated by Western blotting showing PARP cleavage and increased MYOG levels, respectively. RNA-seq suggested that PFI-63 and PFI-90 were KDM inhibitors. In vitro enzymatic inhibition assays confirmed activity against multiple KDMs with most potent inhibition of KDM3B. Western analysis for methylation at H3K4 and H3K9 showed increases after PFI-90 treatment. NMR techniques confirmed biophysical binding of PFI-90 to KDM3B. RNA-seq of KDM knockdowns demonstrated that KDM3B knockdown most closely recapitulated PFI-90’s downregulation of P3F targets. Knockdown of KDM1A recapitulated PFI-90’s upregulation of myogenesis and apoptosis. ChIP-seq analysis showed increased levels of H3K9me2 at P3F sites while H3K4me3 was increased in muscle differentiation and apoptosis. In two different in vivo xenograft models of FP-RMS, PFI-90 treatment delayed tumor progression vs DMSO control. CONCLUSION: We identified novel multi-KDM inhibitors with highest potency for KDM3B. Downregulation of P3F by KDM3B inhibition is associated with increased H3K9me2 at P3F sites. PFI-90 also inhibits KDM1A which increases H3K4me3 at myogenesis and apoptosis genes. Thus, PFI-90 is a novel multi-KDM inhibitor whose biological effect on FP-RMS is by inhibition of KDM3B and KDM1A. Pre-clinical validation via FP-RMS xenograft models showed that PFI-90 delayed tumor progression. PFI-90 thus represents a promising novel compound for the treatment of FP-RMS, and potentially, other transcriptionally driven cancers. Citation Format: Yong Yean Kim, Girma Woldemichael, Berkley Gryder, Silvia Pomella, Ranuka Sinniah, Josh Kowalczyk, Young Song, Mehal Churiwal, Joseph Barchi, John Schneekloth, Xinyu Wen, Hsein-Chao Chou, Barry Okeefe, John Shern, Robert Hawley, Javed Khan. Novel histone lysine demethylase inhibitors disrupt PAX3-FOXO1-driven transcriptional output in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 703.