AbstractThe transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19 µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other “FOXO-resistant” cancers.