Abstract 47: Identification of first-in-class KDM3B inhibitors that suppress PAX3-FOXO1 oncogene activity in fusion positive rhabdomyosarcoma

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 activation of super enhancer driven transcription. Thus, this fusion gene represents a unique vulnerability in FP-RMS which can be targeted by small molecules. MATERIALS AND METHODS: Using luciferase assays simultaneously monitoring PAX3-FOXO1 super enhancer and general transcription activity in RMS cells, 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 characterize the functions of fusion gene inhibitors, histone lysine demethylase (KDM) enzyme inhibition assay and in vitro proliferation assay was performed. Western analysis was used to assess expression level of MYOG, PARP, and PAX3-FOXO1 in RMS cells after treatment with inhibitors. NMR techniques WaterLOGSY and CPMG were used to determine direct binding of compound to KDM3B. In vivo FP-RMS xenograft mouse model was used to determine tumor suppression by PAX3-FOXO1 inhibitors. RESULTS: PAX3-FOXO1 selective cell-based assay identified 64 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, and its analog PFI-90 was identified using a chemical similarity screen based on PFI-63. RNA-seq analysis on RMS cells treated with PFI-63 and PFI-90 indicated these compounds inhibit KDMs, resulting in apoptosis and myogenic differentiation while PAX3-FOXO1 downstream gene sets were repressed. Activation of apoptosis and myogenic differentiation was validated by Western analysis for PARP cleavage and increased expression of MYOG respectively. In vitro enzymatic inhibition assay confirmed activity against KDMs with highest specificity for KDM3B. Western analysis for methylation status of histone 3 lysines at positions K27, K4, and K9 showed increased histone 3 methylation at K4 and K9 sites after treatment. Furthermore, WaterLOGSY and CPMG NMR techniques showed direct binding of PFI-90 to KDM3B. Finally, PFI-90 delayed tumor progression in a FP-RMS xenograft mouse model. CONCLUSION: We identified two KDM3B selective inhibitors which directly bind to KDM3B and disrupt PAX3-FOXO1 downstream targets in FP-RMS. PFI-63 and PFI-90 induce apoptosis and cell differentiation resulting in delayed tumor progression in vivo. Thus, we describe here novel inhibitors of KDM3B that inhibit PAX3-FOXO1 action. These novel compounds represent a potential new therapy for FP-RMS. Citation Format: Yong Yean Kim, Robert G. Hawley, Berkley Gryder, Silvia Pomella, Joshua Kowalczyk, Joseph Barchi, Young Song, Javed Khan. Identification of first-in-class KDM3B inhibitors that suppress PAX3-FOXO1 oncogene activity in fusion positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 47.