RRM2 is a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma

Abstract

SummaryNeuroblastoma 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. 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. In vitro knock down and pharmacological RRM2 inhibition highlight RRM2 dependency in neuroblastoma cells, further supported by the finding that co-overexpression of RRM2 in a dβh-MYCN transgenic 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 revealed HEXIM1 as 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 inhibition in 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.

Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

“…Although earlier studies demonstrated a synergistic effect of triapine with chemotherapeutics or PARP inhibitors in other cancer types (35, 36), we did not observe marked synergy with these drug combinations in EwS cells, which may indicate tumor-specific effects with combinatorial drug applications. Importantly, similar to a very recent report in neuroblastoma (37), we noted a strong synergistic effect of triapine with CHEK1 or WEE1 inhibitors in EwS cells.…”

From DNA Copy Number Gains and Tumor Dependencies to Novel Therapeutic Targets for High-Risk Neuroblastoma

“…Furthermore, a zebrafish MYCN/RRM2 co-overexpression model revealed a strong increase in tumor penetrance compared to MYCN only overexpressing animals. Finally, we demonstrated synergism for combined RRM2 and CHK1 inhibition thus pointing towards this drug vulnerability as an important future angle for therapeutic exploration [125].…”

“…We recently identified the RRM2 gene as target for focal gains and amplifications in high-risk NBs, amongst others resulting from chromothripsis [125]. RRM2 encodes for the 'ribonucleotide reductase regulatory subunit 2 (RRM2) and together with RRM1 forms the RNR ribonucleotide reductase enzyme that ensures dNTP production essential for DNA replication and repair.…”

“…RRM2 has been proposed as driver oncogene in prostate cancer [126] and has been shown to play a critical role in melanoma [127] and Ewing sarcoma [128]. Based on its implication in focal amplifications and gains together with strong upregulation in development of TH-MYCN driven NB in mice and inverse correlation with survival in NB patients, our team further investigated the role of RRM2 through in vitro and in vivo experiments [125]. NB cell lines were shown to strongly depend on RRM2 for survival.…”

“…Both mouse and zebrafish models have been established for MYCN-driven NB formation [22,[46][47][48]50,54,59]. In particular the zebrafish model has proven to be an efficient tool for testing co-dependencies as illustrated by successful studies for multiple candidates including mutant ALK [48], LMO1 [153], LIN28B [59], RRM2 ( [125], p. 2), BIRC5 (Dolman et al, in review) and BRIP1 (Vanhauwaert et al, in preparation). One particular advantage of zebrafish models is access to early developing lesions that can be isolated by FACs sorting [154] and which is currently under further investigation in our lab for deeper exploration of early transforming events using single cell technologies.…”