GSE161900
The dependency factor RRM2 at the nexus of a copy number driven regulatory network and a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma [compound_3AP]
Neuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10-15% of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease at diagnosis despite intensive multi-modal therapies of which nearly 50% relapse and die of their disease. In contrast to the overall paucity of mutations, high-risk neuroblastoma nearly invariably present with recurrent somatic segmental chromosome copy number variants. For several focal aberrations (e.g. MYCN and LIN28B amplification), the direct role in tumor formation has been established. However, for recurrent aberrations, such as chromosome 2p and 17q gains, the identification of genes contributing to tumor initiation or progression has been challenging due to the scarcity of small segmental gains or amplifications. In this study, we identified and functionally evaluated the 'ribonucleotide reductase regulatory subunit 2' (RRM2) as a top-ranked 2p putative co-driver and therapeutic target in high-risk neuroblastoma enforcing replicative stress resistance. RRM2 dependency was shown in vitro through knockdown in neuroblastoma cells, with transcriptome analysis revealing downregulation of pediatric tumor markers. Transgenic zebrafish lines expressing RRM2 in immature sympathoblasts were crossed with a d?h-MYCN transgenic zebrafish line, with RRM2 overexpression dramatically increasing tumor penetrance from 20% to nearly 100%, accelerating neuroblastoma formation with tumors formed as early as 5 weeks of age in the double positive fish.  Through CasID, we aimed to identify RRM2 promotor bound proteins. and enriched amongst others HEXIM1 and the NurRD complex. Importantly, adaptive responses regulating RRM2 expression are also controlled by ATR/CHK1 and WEE1 cell cycle checkpoint kinases, as shown by pharmacological inhibition with small molecule inhibitors. We provide in vitro and in vivo data showing that RRM2 inhibition with triapine strongly sensitizes neuroblastoma cells to CHK1 inhibition and that combined treatment could robustly induce upregulated PD-L1 surface expression. Altogether, we propose RRM2 as a bona fide target to explore novel synergistic drug combinations through phase I clinical trials. Overall design: Human neuroblastoma cell lines SHSY5Y, SK-N-BE(2)-C IMR32 and CLBGA were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 IU/mL penicillin/streptomycin and 2mM L- Glutamine. All cell lines were grown at 37°C and 5% CO2. TThe adherent cell lines were seeded in a T-75 flask at density of 2x106 – 2.25x106 cells per flask, depending on the cell line. Cells were allowed to adhere for 48h, after which the medium was replaced by fresh medium and 3AP (or DMSO) was added. After 48h of treatment the cells were scraped, centrifuged, for 5 minutes at 1200 rpm, and the pellet was washed twice with ice-cold PBS.