KDM3B inhibitors disrupt PAX3-FOXO1 oncogenic activity in fusion positive rhabdomyosarcoma

fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 com- pounds using an engineered cell line that monitors PAX3-FOXO1 transcrip- tional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethy- lases (KDMs) as its targets. enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phe- nocopies P3FI-90 effects. thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. their potent suppression of PAX3- FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.