RRM2 is a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma
Authors: Nunes Carolina , Depestel Lisa , Mus Liselot , Keller Kaylee , Delhaye Louis , Louwagie Amber , Rishfi Muhammad , Dolman Emmy , Olexiouk Volodimir , Bartenhagen Christoph , De Vloed Fanny , Sanders Ellen , Eggermont Aline , Van Laere Jolien , Desmet Els , Van Loocke Wouter , Morscio Julie , Loontiens Siebe , Depuydt Pauline , Decaesteker Bieke , Tilleman Laurentijn , Van Nieuwerburgh Filip , Deforce Dieter , De Wilde Bram , Van Vlierberghe Pieter , Vermeirssen Vanessa , Goossens Steven , Eyckerman Sven , Van Neste Christophe , Roberts Stephen , Fischer Matthias , Molenaar Jan , Durinck Kaat , Speleman Frank 
Published: November 2020

Neuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10-15 percent of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease at diagnosis. Despite intensive multi-modal therapies nearly 50 percent of high-risk cases 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. MYCNandLIN28Bamplification), 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.In vitroknock down and pharmacological RRM2 inhibition highlight RRM2 dependency in neuroblastoma cells, further supported by the finding that co-overexpression of RRM2 in adβh-MYCNtransgenic zebrafish line increased tumor penetrance with 80% and accelerated tumor formation. Given the critical role of RRM2 in replication fork progression and regulation of RRM2 through ATR/CHK1 signaling, we tested combined RRM2 and ATR/CHK1 small molecule inhibition with triapine and BAY1895344/prexasertib respectively, and observed strong synergism, in particular for combined RRM2 and CHK1 inhibition. Transcriptome analysis following combinatorial drugging revealedHEXIM1as one of the strongest upregulated genes. Using programmable DNA binding of dCas9 with a promiscuous biotin ligase, RRM2 promotor bound proteins were identified including HEXIM1 and NurRD complex members, supporting a cooperative role for HEXIM1 upregulation together with CHK1 inhibition in further attenuating RRM2 expression levels. We evaluated the impact of combined RRM2/CHK1 inhibitionin vivo, with treatment of a murine xenograft model showing rapid and complete tumor regression, without tumor regrowth upon treatment arrest. In conclusion, we identified RRM2 as a novel dependency gene in neuroblastoma and promising target for synergistic drug combinations with small compounds targeting DNA checkpoint regulators.