Neuroblastoma is a pediatric tumor of the sympathetic nervous system associated with poor survival in high-risk cases. Current options for targeted therapy for high-risk neuroblastoma patients are limited and single compound strategies almost invariably fail due to escape mechanisms, driven either by initial tumor heterogeneity or through adaptive (epigenetic) responses or mutations. Consortia such as SIOPEN and ITCC (www.itcc-consortium.org) explore novel therapies in neuroblastoma patients through phase I/II clinical (basket) trials. However, parallel intensive research programs to identify novel therapeutic vulnerabilities and drug targeting strategies are of utmost importance to fuel these initiatives and help prioritize the most potent drug combinations, with limited toxicity. While several important chemotherapeutic compounds work by increasing replication stress in cancer cells to toxic levels, more recent and putatively less toxic small molecule-based approaches are now emerging that directly target the DNA damage response (DDR) pathways such as ATR, CHK1 and PARP inhibitors. The ATR checkpoint serves to protect cells from replication fork collapse and promotes fork restart under conditions of replicative stress. Whereas initially stalled forks can undergo repair through replication fork reversal, prolonged fork stalling will eventually result in fork collapse characterized by the formation of double strand breaks (DSBs). Loss-of-function mutations in BRCA1 have a well-defined role in tumor initiation through enhanced genomic instability and can be therapeutically exploited through synthetic lethal interaction with PARP1 inhibition in BRCA1-deficient cancers. A similar dependence to compensatory DNA damage repair (DDR) pathways through mutations in other genes that protect genomic integrity has been proposed and is referred to as the BRCAness phenotype. Cancers undergoing high replicative stress levels, such as neuroblastoma, typically exhibit high expression levels of DDR proteins such as BRCA1 and RAD51, likely resulting from elevated ATR-CHK1 activation levels. In this context, it is of interest that ‘cyclin dependent kinase 12’ (CDK12) has been shown to play an instrumental role in control of expression levels of key DDR proteins involved in repair of DSBs at collapsed forks and maintaining genomic integrity in rapidly dividing cancer cells. In line with these findings, mutational inactivation of CDK12 was observed in ovarian and prostate cancer, imposing a BRCAness phenotype which predicts sensitivity to targeted treatments such as PARP1 inhibition. Given the above-mentioned ATR-CHK1 activation mediated upregulation of DNA repair factors in neuroblastoma, we hypothesize that CDK12 inhibition could impose a BRCAness phenotype and could thus be a route to synthetic lethality in neuroblastoma. In this project, we first pursue an in-depth preclinical evaluation of pharmacological CDK12 intervention as an entry point for synergistic drug interactions in neuroblastoma. To this end, we performed a series of combination drug screens which led to the identification of drug synergism between pharmacological CDK12 inhibition and a robust set of clinically validated agents. In this project, we will evaluate in-depth the most potent drug combinations (“pick-the-winner” strategy) in vivo using patient--derived xenograftzebrafish and murine models to prepare a pre-clinical data package ready for testing in a novel clinical trial.