Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system1. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2–4. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type1,2,5. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

The broad clinical spectrum of neuroblastoma ranges from spontaneous regression to rapid progression despite intensive multimodal therapy. This diversity is not fully explained by known genetic aberrations, suggesting the possibility of epigenetic involvement in pathogenesis. In pursuit of this hypothesis, we took an integrative approach to analyze the methylomes, transcriptomes, and copy number variations in 105 cases of neuroblastoma, complemented by primary tumor- and cell line-derived global histone modification analyses and epigenetic drug treatment in vitro We found that DNA methylation patterns identify divergent patient subgroups with respect to survival and clinicobiologic variables, including amplified MYCN Transcriptome integration and histone modification-based definition of enhancer elements revealed intragenic enhancer methylation as a mechanism for high-risk-associated transcriptional deregulation. Furthermore, in high-risk neuroblastomas, we obtained evidence for cooperation between PRC2 activity and DNA methylation in blocking tumor-suppressive differentiation programs. Notably, these programs could be re-activated by combination treatments, which targeted both PRC2 and DNA methylation. Overall, our results illuminate how epigenetic deregulation contributes to neuroblastoma pathogenesis, with novel implications for its diagnosis and therapy. Cancer Res; 76(18); 5523-37. ©2016 AACR.

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Background Sodium glucose co-transporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in diverse patient populations, but their mechanism of action requires further study. Aims To explore the effect of empagliflozin on circulating levels of intracellular proteins in patients with heart failure, using large-scale proteomics. Methods Over 1250 circulating proteins were measured at baseline, week 12 and week 52 in 1134 patients from EMPEROR-Reduced and EMPEROR-Preserved, using the Olink® Explore 1536 platform. Statistical and bioinformatical analyses identified differentially expressed proteins (empagliflozin vs placebo), which were then linked to demonstrated biological actions in the heart and kidneys. Results At week 12, 32 of 1283 proteins fulfilled our threshold for being differentially expressed, i.e., their levels were changed by ≥10% with a false discovery rate < 1% (empagliflozin vs placebo). Among these, nine proteins demonstrated the largest treatment effect of empagliflozin: insulin-like growth factor-binding protein 1, transferrin receptor protein 1, carbonic anhydrase 2, erythropoietin, protein-glutamine gamma-glutamyltransferase 2, thymosin beta-10, U-type mitochondrial creatine kinase, insulin-like growth factor-binding protein 4, and adipocyte fatty acid-binding protein 4. The changes of the proteins from baseline to week 52 were generally concordant with the changes from baseline to week 12, except empagliflozin reduced levels of kidney injury molecule-1 by ≥10% at week 52, but not at week 12. The most common biological action of differentially-expressed proteins appeared to be the promotion of autophagic flux in the heart, kidney or endothelium, a feature of 6 proteins. Other effects of differentially-expressed proteins on the heart included the reduction of oxidative stress, inhibition of inflammation and fibrosis, and the enhancement of mitochondrial health and energy, repair and regenerative capacity. The actions of differentially expressed proteins in the kidney involved promotion of autophagy, integrity and regeneration, suppression of renal inflammation and fibrosis, and modulation of renal tubular sodium reabsorption. Conclusions Changes in circulating protein levels in patients with heart failure are consistent with the findings of experimental studies that have shown that the effects of SGLT2 inhibitors are likely related to actions on the heart and kidney to promote autophagic flux, nutrient deprivation signaling and transmembrane sodium transport.