RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition

Abstract

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

The fork restart factor PHF6 interacts with RRM2 and binds to H3K56ac marked nascent DNA

“…Protein-protein interactions are increasingly recognized as an emerging drug modality and therefore we propose that pharmacological targeting PHF6-RRM2 is a promising therapeutic approach in cancer 62 . Given that we previously identified RRM2 as key replicative stress resistor in the context of neuroblastoma 26 , we aimed in this study to functionally evaluate the role of PHF6 in this tumor entity. Through CUT&RUN analyses we could show for the first time that genome-wide binding sites of PHF6 significantly overlap with those for previously described neuroblastoma specific adrenergic core regulatory circuitry (CRC) factors including MYCN, HAND2, PHOX2B, GATA3 and ISL1.…”

Chromosome 11q loss and MYCN amplification demonstrate synthetic lethality with checkpoint kinase 1 inhibition in neuroblastoma

“…Independently, it has been shown that MNA is also capable of inducing the accumulation replication stress by activating dormant origins of replication (27,35). Despite being very different genomic aberrations, these studies have shown that increased replication stress associated with 11q loss or MNA induces increased sensitivity to inhibition of key replication stress proteins (27,(34)(35)(36)(37)(38). As CHK1 is essential to the replication stress response via its role in replication fork stabilization and modulation of the S-phase and G 2 -M cell cycle checkpoints during DNA damage repair, it is perhaps unsurprising that we observe effective cell killing when CHK1 is inhibited in cells with genomic aberrations that are known to cause additional replication stress.…”

“…Previous studies have shown that 11q loss induces in haploinsufficiency of key DNA damage repair genes, which results in the accumulation of DNA damage and thus, replication stress ( 34 ). Independently, it has been shown that MNA is also capable of inducing the accumulation replication stress by activating dormant origins of replication ( 27 , 35 ). Despite being very different genomic aberrations, these studies have shown that increased replication stress associated with 11q loss or MNA induces increased sensitivity to inhibition of key replication stress proteins ( 27 , 34 – 38 ).…”

“…As CHK1 is essential to the replication stress response via its role in replication fork stabilization and modulation of the S-phase and G 2 -M cell cycle checkpoints during DNA damage repair, it is perhaps unsurprising that we observe effective cell killing when CHK1 is inhibited in cells with genomic aberrations that are known to cause additional replication stress. The dependency of NB cells on replication stress response pathways for survival is further elucidated in a recently published study where it is demonstrated that MNA-driven NB is resistant to replication stress via overexpression of ribonucleotide reductase subunit M2 (RRM2) ( 35 ).…”

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

“…However, RRM1 and RRM2 may represent partial exceptions to this, which we identified among the top hits sensitizing to etoposide and vincristine. Recently, combined RRM2 and CHK1 inhibition was shown to be synergistic and non-toxic in neuroblastoma xenografts owing to replicative stress due to stalled replication forks 58 , a process in which TOP2, the target of etoposide, also plays a key role 59 .…”