KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 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 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (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 phenocopies 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.there is lack of therapies targeting the PAX3-FOXO1 fusion oncogene in fusion-positive rhabdomyosarcoma (FP-RMS). here, the authors identify and characterise an inhibitor with highest inhibition of histone lysine demethylase 3B that suppresses PAX3-FOXO1 activity in FP-RMS.