ABSTRACT Combination chemotherapy is crucial for achieving durable cancer cures, however, developing safe and effective drug combinations has been a significant challenge. To improve this process, we conducted large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritized neuroblastoma, the most common pediatric solid tumor, where 50% of high-risk patients do not survive. Our screen examined all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest drug combination screens ever reported. Remarkably, using dense drug-drug rescreening, we found that the top CRISPR-nominated drug combinations were far more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discovered that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using PDX models. Our findings provide a valuable resource for the development of improved chemotherapeutic strategies and demonstrate the feasibility of using targeted CRISPR knockout to discover new combinations with common chemotherapeutics, a methodology with application across all cancers.

A CRISPR-drug perturbational map for identifying new compounds to combine with commonly used chemotherapeutics