Supplementary MaterialsSupplemental material 41419_2018_924_MOESM1_ESM. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is usually highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA AC220 price damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 AC220 price knockdown or its pharmacological inhibitor induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors. Introduction MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, which has essential roles in detecting and fixing DNA double-strand breaks (DSBs) and activation of the DNA damage response (DDR) via ATM1,2. Within the complex, the NBS1 and RAD50 moieties mediate nuclear localization and interactions with DNA and protein partners. MRE11 is essential to stabilize the complex allowing its accumulation, and to provide the nuclease activities required for the resection of the broken DNA ends3,4. Hypomorphic MRE11 mutations are responsible for the inherited Ataxia-Telangiectasia-like disorder (ATLD), which shares cellular and clinical phenotypes (including immunodeficiency, sterility, and radiosensitivity) with Ataxia Telangiectasia (A-T) and Nijmegen breakage syndrome (NBS), caused by mutations in the ATM and NBS1 genes, respectively5,6. Total loss of prospects to early embryonic lethality due to severe proliferation defects in vertebrate cells7C10. Appropriate animal models recapitulate the main features of human syndromes and support MRN tumor suppressive function11C13, consistent with the increased cancer susceptibility observed in MRN-defective human syndromes. Much like other DNA repair proteins, MRE11 also plays a pivotal role in controlling the integrity of DNA replication, preventing the deleterious effects of replication stress (RS)14C17. Indeed, an inefficient response to RS seems to contribute to the genesis of developmental disorders of the nervous system, in patients and animal models transporting mutations in MRN genes18,19. MYCN is usually a member of the MYC family of transcription factors, largely expressed in, and required for, nervous system development20. As an oncogene, it is deregulated in several neuronal and non-neuronal tumors of child years, including neuroblastoma, medulloblastoma, retinoblastoma, astrocytoma, rhabdomyosarcoma, Wilms tumor, and in adulthood tumors, such as non-small cell lung malignancy and breast malignancy (http://www.cancerindex.org/geneweb/MYCN.htm). At least in neuroblastoma, where patients are typically stratified into risk groups based on multiple parameters, amplification (MNA) represents the most relevant AC220 price and impartial negative prognostic factor allowing straightforward patient classification into the high-risk group21C23. Despite intense multimodal treatment, MNA neuroblastoma patients often relapse and succumb to their disease22, which underscores the need for more Tpo effective therapeutic methods for these children. MYC proteins promote RS, DNA damage, and DDR by several mechanisms24C31. Increased levels of RS have been clearly detected in main MNA tumors as compared to MYCN single copy (MNSC) samples31. Moreover, DNA repair is among the most significantly deregulated gene ontology groups in neuroblastomas sharing a MYCN signature32. Overall, these data suggest that coping with RS and DNA damage is cogent in these tumors and they are consistent with the knowledge that DDR proteins can be recruited by oncogenes to dampen oncogene-dependent RS, eventually favoring cancer cell survival33C36. We recently showed the MRE11, RAD50, and NBS1 are transcriptionally regulated by MYCN in order to prevent the accumulation of RS-dependent DNA damage during MYCN-driven expansion of cerebellar granule progenitor cells26. Whether the MRN complex is essential to prevent the deleterious effects of MYCN-dependent RS also in cancer cells was poorly investigated, so far. Here, we explored the involvement of MRE11 in neuroblastoma as a model for MYCN-driven tumors and addressed the possibility to target the MRN complex to trigger intolerable levels of RS-dependent DNA damage in MNA/high-risk tumors. Results MRE11 is overexpressed in MNA neuroblastoma and is essential for MYCN-dependent proliferation By interrogating multiple neuroblastoma gene expression datasets on the R2-Genomics platform (http://r2.amc.nl), we noticed that very high MRE11 expression was associated with reduced overall survival in primary human neuroblastoma (Fig.?1a and S1). Consistently, MRE11 mRNA expression was significantly higher in worst prognosis cases characterized by MYCN-amplified (MNA) compared to MNSC neuroblastoma tumors 1 (Fig.?1b). This was further confirmed in neuroblastoma cell lines, at the RNA and protein levels (Fig.?1c). Notably, a very low MRE11 expression was instead associated with.