DHODH is an independent prognostic marker and potent therapeutic target in neuroblastoma

Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.

iodide (P3566, Thermo Fisher Scientific). Data were acquired using a BD LSRFortessa system (BD Biosciences) and analyzed with FlowJo v10 software.

ChIP library preparation and sequencing
Tissue for ChIP sequencing was snap frozen and stored in liquid nitrogen ChIP experiments and library preparation was performed by Active Motif using the HistonePath workflow.
Mouse tumor tissues were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was then isolated by adding lysis buffer followed by tissue disruption with a Dounce homogenizer. Lysates were sonicated and DNA sheared to an average length of 300-500 bp. Genomic DNA (input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. DNA was quantified on a NanoDrop spectrophotometer. 30 µg chromatin was precleared with protein A agarose beads (Invitrogen) and genomic DNA regions of interest isolated using 4 µg of H3K27Ac antibody (Active Motif, polyclonal, catalog number 39133). Complexes were washed, eluted with SDS buffer, and subjected to RNase and proteinase K treatment.
Crosslinks were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR reactions were carried out in triplicates on specific genomic regions using SYBR Green Supermix (Bio-Rad).
Illumina sequencing libraries were prepared from ChIP and input DNA by end-polishing, dAaddition and adaptor ligation. After PCR amplification, DNA libraries were sequenced on the NextSeq 500 platform (Illumina), 75 bp reads, single-end.