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Outcomes for children with relapsed and refractory neuroblastoma are dismal. The combination of irinotecan and temozolomide has activity in these patients, and its acceptable toxicity profile makes it an excellent backbone for study of new agents. We aimed to test the addition of temsirolimus or dinutuximab to irinotecan–temozolomide in patients with relapsed or refractory neuroblastoma. For this open-label, randomised, phase 2 selection design trial of the Children's Oncology Group (COG; ANBL1221), patients had to have histological verification of neuroblastoma or ganglioneuroblastoma at diagnosis or have tumour cells in bone marrow with increased urinary catecholamine concentrations at diagnosis. Patients of any age were eligible at first designation of relapse or progression, or first designation of refractory disease, provided organ function requirements were met. Patients previously treated for refractory or relapsed disease were ineligible. Computer-based randomisation with sequence generation defined by permuted block randomisation (block size two) was used to randomly assign patients (1:1) to irinotecan and temozolomide plus either temsirolimus or dinutuximab, stratified by disease category, previous exposure to anti-GD2 antibody therapy, and tumour MYCN amplification status. Patients in both groups received oral temozolomide (100 mg/m2 per dose) and intravenous irinotecan (50 mg/m2 per dose) on days 1–5 of 21-day cycles. Patients in the temsirolimus group also received intravenous temsirolimus (35 mg/m2 per dose) on days 1 and 8, whereas those in the dinutuximab group received intravenous dinutuximab (17·5 mg/m2 per day or 25 mg/m2 per day) on days 2–5 plus granulocyte macrophage colony-stimulating factor (250 μg/m2 per dose) subcutaneously on days 6–12. Patients were given up to a maximum of 17 cycles of treatment. The primary endpoint was the proportion of patients achieving an objective (complete or partial) response by central review after six cycles of treatment, analysed by intention to treat. Patients, families, and those administering treatment were aware of group assignment. This study is registered with ClinicalTrials.gov, number NCT01767194, and follow-up of the initial cohort is ongoing. Between Feb 22, 2013, and March 23, 2015, 36 patients from 27 COG member institutions were enrolled on this groupwide study. One patient was ineligible (alanine aminotransferase concentration was above the required range). Of the remaining 35 patients, 18 were randomly assigned to irinotecan–temozolomide–temsirolimus and 17 to irinotecan–temozolomide–dinutuximab. Median follow-up was 1·26 years (IQR 0·68–1·61) among all eligible participants. Of the 18 patients assigned to irinotecan–temozolomide–temsirolimus, one patient (6%; 95% CI 0·0–16·1) achieved a partial response. Of the 17 patients assigned to irinotecan–temozolomide–dinutuximab, nine (53%; 95% CI 29·2–76·7) had objective responses, including four partial responses and five complete responses. The most common grade 3 or worse adverse events in the temsirolimus group were neutropenia (eight [44%] of 18 patients), anaemia (six [33%]), thrombocytopenia (five [28%]), increased alanine aminotransferase (five [28%]), and hypokalaemia (four [22%]). One of the 17 patients assigned to the dinutuximab group refused treatment after randomisation; the most common grade 3 or worse adverse events in the remaining 16 patients evaluable for safety were pain (seven [44%] of 16), hypokalaemia (six [38%]), neutropenia (four [25%]), thrombocytopenia (four [25%]), anaemia (four [25%]), fever and infection (four [25%]), and hypoxia (four [25%]); one patient had grade 4 hypoxia related to therapy that met protocol-defined criteria for unacceptable toxicity. No deaths attributed to protocol therapy occurred. Irinotecan–temozolomide–dinutuximab met protocol-defined criteria for selection as the combination meriting further study whereas irinotecan–temozolomide–temsirolimus did not. Irinotecan–temozolomide–dinutuximab shows notable anti-tumour activity in patients with relapsed or refractory neuroblastoma. Further evaluation of biomarkers in a larger cohort of patients might identify those most likely to respond to this chemoimmunotherapeutic regimen. National Cancer Institute.

The HIT-SIOP-PNET4 randomised trial for standard risk medulloblastoma (MB) (2001–2006) included 338 patients and compared hyperfractionated and conventional radiotherapy. We here report the long-term outcome after a median follow up of 7.8 years, including detailed information on relapse and the treatment of relapse. Data were extracted from the HIT Group Relapsed MB database and by way of a specific case report form. The event-free and overall (OS) survival at 10 years were 76 ± 2 % and 78 ± 2 % respectively with no significant difference between the treatment arms. Seventy-two relapses and three second malignant neoplasms were reported. Thirteen relapses (18 %) were isolated local relapses in the posterior fossa (PF) and 59 (82 %) were craniospinal, metastatic relapses (isolated or multiple) with or without concurrent PF disease. Isolated PF relapse vs all other relapses occurred at mean/median of 38/35 and 28/26 months respectively (p = 0.24). Late relapse, i.e. >5 years from diagnosis, occurred in six patients (8 %). Relapse treatment consisted of combinations of surgery (25 %), focal radiotherapy (RT 22 %), high dose chemotherapy with stem cell rescue (HDSCR 21 %) and conventional chemotherapy (90 %). OS at 5 years after relapse was 6.0 ± 4 %. In multivariate analysis; isolated relapse in PF, and surgery were significantly associated with prolonged survival whereas RT and HDSCR were not. Survival after relapse was not related to biological factors and was very poor despite several patients receiving intensive treatments. Exploration of new drugs is warranted, preferably based on tumour biology from biopsy of the relapsed tumour.

Background Although several studies have been conducted on the role of surgery in localized neuroblastoma, the impact of surgical timing and extent of primary tumor resection on outcome in high-risk patients remains controversial. Methods Patients from the German neuroblastoma trial NB97 with localized neuroblastoma INSS stage 1–3 age > 18 months were included for retrospective analysis. Imaging reports were reviewed by two independent physicians for Image Defined Risk Factors (IDRF). Operation notes and corresponding imaging reports were analyzed for surgical radicality. The extent of tumor resection was classified as complete resection (95–100%), gross total resection (90–95%), incomplete resection (50–90%), and biopsy (<50%) and correlated with local control rate and outcome. Patients were stratified according to the International Neuroblastoma Risk Group (INRG) staging system. Survival curves were estimated according to the method of Kaplan and Meier and compared by the log-rank test. Results A total of 179 patients were included in this study. 77 patients underwent more than one primary tumor operation. After best surgery, 68.7% of patients achieved complete resection of the primary tumor, 16.8% gross total resection, 14.0% incomplete surgery, and 0.5% biopsy only. The cumulative complication rate was 20.3% and the surgery associated mortality rate was 1.1%. Image defined risk factors (IDRF) predicted the extent of resection. Patients with complete resection had a better local-progression-free survival (LPFS), event-free survival (EFS) and OS (overall survival) than the other groups. Subgroup analyses showed better EFS, LPFS and OS for patients with complete resection in INRG high-risk patients. Multivariable analyses revealed resection (complete vs. other), and MYCN (non-amplified vs. amplified) as independent prognostic factors for EFS, LPFS and OS. Conclusions In patients with localized neuroblastoma age 18 months or older, especially in INRG high-risk patients harboring MYCN amplification, extended surgery of the primary tumor site improved local control rate and survival with an acceptable risk of complications.

Dysregulation and enhanced expression of MYC transcription factors (TFs) including MYC and MYCN contribute to the majority of human cancers. For example, MYCN is amplified up to several hundredfold in high-risk neuroblastoma. The resulting overexpression of N-myc aberrantly activates genes that are not activated at low N-myc levels and drives cell proliferation. Whether increasing N-myc levels simply mediates binding to lower-affinity binding sites in the genome or fundamentally changes the activation process remains unclear. One such activation mechanism that could become important above threshold levels of N-myc is the formation of aberrant transcriptional condensates through phase separation. Phase separation has recently been linked to transcriptional regulation, but the extent to which it contributes to gene activation remains an open question. Here we characterized the phase behavior of N-myc and showed that it can form dynamic condensates that have transcriptional hallmarks. We tested the role of phase separation in N-myc-regulated transcription by using a chemogenetic tool that allowed us to compare non-phase-separated and phase-separated conditions at equivalent N-myc levels, both of which showed a strong impact on gene expression compared to no N-myc expression. Interestingly, we discovered that only a small percentage (<3%) of N-myc-regulated genes is further modulated by phase separation but that these events include the activation of key oncogenes and the repression of tumor suppressors. Indeed, phase separation increases cell proliferation, corroborating the biological effects of the transcriptional changes. However, our results also show that >97% of N-myc-regulated genes are not affected by N-myc phase separation, demonstrating that soluble complexes of TFs with the transcriptional machinery are sufficient to activate transcription. Oncoprotein transcription factor MYC undergoes phase separation, forming transcriptionally active condensates. The chemogenetic tool SPARK-ON reveals that MYC phase separation selectively modulates the transcriptome and promotes cell proliferation.

Oncoproteins of the MYC family are major drivers of human tumorigenesis. Since a large body of evidence indicates that MYC proteins are transcription factors, studying their function has focused on the biology of their target genes. Detailed studies of MYC-dependent changes in RNA levels have provided contrasting models of the oncogenic activity of MYC proteins through either enhancing or repressing the expression of specific target genes, or as global amplifiers of transcription. In this Review, we first summarize the biochemistry of MYC proteins and what is known (or is unclear) about the MYC target genes. We then discuss recent progress in defining the interactomes of MYC and MYCN and how this information affects central concepts of MYC biology, focusing on mechanisms by which MYC proteins modulate transcription. MYC proteins promote transcription termination upon stalling of RNA polymerase II, and we propose that this mechanism enhances the stress resilience of basal transcription. Furthermore, MYC proteins coordinate transcription elongation with DNA replication and cell cycle progression. Finally, we argue that the mechanism by which MYC proteins regulate the transcription machinery is likely to promote tumorigenesis independently of global or relative changes in the expression of their target genes.The MYC oncoproteins are transcription factors, but the molecular mechanism of their oncogenic activity is unclear. MYC proteins promote transcription termination in stress conditions, which is proposed to increase cellular resilience to stress and to promote tumorigenesis independently of changes in the expression of their target genes.

International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0–16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3–16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant; n=65) and childhood patients (≥4·3 years; MBSHH-Child; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR [n=65] and MBGrp4-HR [n=85]) and low-risk (MBGrp3-LR [n=50] and MBGrp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82–100]); standard risk (50 [23%] patients; 81% survival [70–94]); high-risk (82 [38%] patients; 42% survival [31–56]); and very high-risk (29 [13%] patients; 28% survival [14–56]). The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18 , while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors. PARP inhibitors are mainly used to treat BRCA1/2 mutated cancers. Here, the authors show that MYC amplified glioblastomas are sensitive to PARP inhibition due to CDK18 repression, which impairs ATR regulated homologous recombination repair, and that ATR inhibition sensitises glioblastomas to PARP inhibition.

MYC and MYCN oncogenes are frequently upregulated in human liver cancers, yet their functional differences remain unclear. We used a mouse model of intrahepatic cholangiocarcinoma (CCA), constructed by transposon‐mediated somatic gene integration of AKT and YAP into hepatocytes, to investigate the effects of additional integration of Myc or Mycn. Both Myc and Mycn induced a poorly differentiated hepatocellular carcinoma (HCC) component, resulting in the formation of combined hepatocellular‐cholangiocarcinoma (cHCC‐CCA). Interestingly, the ratio of HCC to CCA components differed significantly; AKT/YAP/Mycn (AYN) tumors exhibited a lower proportion of CCA components than AKT/YAP/Myc (AYM) tumors. To explore the underlying mechanisms, we analyzed the expression levels of genes involved in liver differentiation. We found that AYN tumors, at both the mRNA and protein levels, exhibited lower expression of HNF1B, a transcription factor that is highly expressed in human CCA but not in HCC. When Hnf1b was co‐introduced with AYN, the percentage of the CCA area increased significantly. Furthermore, these tumors exhibited increased expression of TEAD proteins, which interact with YAP to initiate transcription. Notably, treatment with a YAP‐TEAD inhibitor suppressed AKT/YAP/Mycn/Hnf1b tumor growth. These findings indicate that Myc and Mycn play distinct roles in the phenotypic determination of primary liver tumors and suggest that their differential effects on Hnf1b expression and subsequent TEAD activation may be a key regulatory mechanism. Combined hepatocellular–cholangiocarcinoma (cHCC‐CCA) is a tumor consisting of a mixture of hepatocellular carcinoma and intrahepatic cholangiocarcinoma (CCA) components, but the mechanisms of development are poorly understood. The activation of Myc may be implicated in the formation of cHCC‐CCA and this work demonstrates for the first time that Myc and Mycn play similar yet distinct roles in liver carcinogenesis, particularly in terms of HNF1B activation, which affects CCA differentiation in hepatocyte‐derived liver cancer. Furthermore, the expression of TEAD proteins, which interact with the YAP protein to initiate transcription, was increased in mouse and human HNF1B‐activated tumors, and since liver tumors did not fully develop in mice treated with a YAP‐TEAD inhibitor, inhibition of this pathway may be an effective therapeutic target for cHCC‐CCA.

Amplification of the locus encoding the oncogenic transcription factor MYCN is a defining feature of high-risk neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of MYCN perturbation in neuroblastoma. At oncogenic levels, MYCN associates with E-box binding motifs in an affinity-dependent manner, binding to strong canonical E-boxes at promoters and invading abundant weaker non-canonical E-boxes clustered at enhancers. Loss of MYCN leads to a global reduction in transcription, which is most pronounced at MYCN target genes with the greatest enhancer occupancy. These highly occupied MYCN target genes show tissue-specific expression and are linked to poor patient survival. The activity of genes with MYCN-occupied enhancers is dependent on the tissue-specific transcription factor TWIST1, which co-occupies enhancers with MYCN and is required for MYCN-dependent proliferation. These data implicate tissue-specific enhancers in defining often highly tumor-specific ‘MYC target gene signatures’ and identify disruption of the MYCN enhancer regulatory axis as a promising therapeutic strategy in neuroblastoma. This study investigates the effects of MYCN on the chromatin and transcriptional landscape of neuroblastoma. The authors find that, at oncogenic levels, MYCN binds to canonical E-boxes at promoters and invades enhancers, leading to transcriptional amplification.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX–histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma. In most cancers, mutations that lead to oncogene activation and tumor suppressor inactivation synergize to promote tumorigenesis. However, in neuroblastomas, MYCN amplification and ATRX mutations are mutually exclusive and incompatible.