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Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine. Pre-clinical cell culture models capturing the heterogeneity of childhood kidney tumours are limited. Here, the authors establish and characterise an organoid biobank of tumour and matched normal organoid cultures from over 50 children with different subtypes of kidney cancer.

CRISPR gene therapy holds the potential to cure a variety of genetic diseases by causing a targeted DNA break, which is repaired by host DNA damage responses. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are more likely to be erroneously repaired by the non-homologous end joining (NHEJ) pathway, which may introduce random insertions or deletions at the cut site. We screened a small library of oncological drug compounds to steer the DNA repair process towards preferential HDR activation. We included forty compounds in the screen based on their mechanism of action. After optimizing the toxicity and adding these compounds during gene editing, nine showed a potential benefit for HDR activation. Three were shown to be beneficial after validation: rucaparib, belinostat and alisertib. The Aurora Kinase A inhibitor alisertib in particular led to an over 4-fold increase in preferential gene correction over gene knock-out in two cell models (HEK293T and Hepa 1–6) at sub-micromolar dosages on the eGFP locus, prompting further validation. On the long term this pathway did show cytotoxicity especially in the HEK293T cells, indicating further mechanistic investigation is needed, but this toxicity was less pronounced in primary hepatocytes.

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.

Over the last years, various precision medicine programs have been developed for pediatric patients with high-risk, relapsed, or refractory malignancies, selecting patients for targeted treatment through comprehensive molecular profiling. In this review, we describe characteristics of these initiatives, demonstrating the feasibility and potential of molecular-driven precision medicine. Actionable events are identified in a significant subset of patients, although comparing results is complicated due to the lack of a standardized definition of actionable alterations and the different molecular profiling strategies used. The first biomarker-driven trials for childhood cancer have been initiated, but until now the effect of precision medicine on clinical outcome has only been reported for a small number of patients, demonstrating clinical benefit in some. Future perspectives include the incorporation of novel approaches such as liquid biopsies and immune monitoring as well as innovative collaborative trial design including combination strategies, and the development of agents specifically targeting aberrations in childhood malignancies.

Hepatoblastoma, the most prevalent pediatric liver cancer, almost always carries a WNT-activating CTNNB1 mutation, yet exhibits notable molecular heterogeneity. To characterize this heterogeneity and identify novel targeted therapies, we perform comprehensive analysis of hepatoblastomas and tumor-derived organoids using single-cell RNA-seq/ATAC-seq, spatial transcriptomics, and high-throughput drug profiling. We identify two distinct tumor epithelial signatures: hepatic ‘fetal’ and WNT-high ‘embryonal’, displaying divergent WNT signaling patterns. The fetal group is enriched for liver-specific WNT targets, while the embryonal group is enriched in canonical WNT target genes. Gene regulatory network analysis reveals enrichment of regulons related to hepatic functions such as bile acid, lipid and xenobiotic metabolism in the fetal subtype but not in the embryonal subtype. In addition, the dichotomous expression pattern of the transcription factors HNF4A and LEF1 allows for a clear distinction between the fetal and embryonal tumor cells. We also perform high-throughput drug screening using patient-derived tumor organoids and identify sensitivity to HDAC inhibitors. Intriguingly, embryonal and fetal tumor organoids are sensitive to FGFR and EGFR inhibitors, respectively, indicating a dependency on EGF/FGF signaling in hepatoblastoma tumorigenesis. In summary, our data uncover the molecular and drug sensitivity landscapes of hepatoblastoma and pave the way for the development of targeted therapies. WNT-activating mutations in CTNNB1 are common in hepatoblastoma, but downstream molecular phenotypes are heterogenous. Using multiomic approaches, the authors identify distinct subgroups of hepatoblastoma cells based on WNT-signaling patterns and create a biobank of patient-derived hepatoblastoma organoids representing these subtypes.

Despite their indisputable importance in neuroblastoma (NB) pathology, knowledge of the bases of NB plasticity and heterogeneity remains incomplete. They may be rooted in developmental trajectories of their lineage of origin, the sympatho-adrenal neural crest. We find that implanting human NB cells in the neural crest of the avian embryo allows recapitulating the metastatic sequence until bone marrow involvement. Using deep single cell RNA sequencing, we characterize transcriptome states of NB cells and their dynamics over time and space, and compare them to those of fetal sympatho-adrenal tissues and patient tumors and bone marrow samples. Here we report remarkable transcriptomic proximities restricted to an early sympathetic neuroblast branch that co-exist with phenotypical adaptations over disease progression and recapitulate intratumor and interpatient heterogeneity. Combining avian and patient datasets, we identify a list of genes upregulated during bone marrow involvement and associated with growth dependency, validating the relevance of our multimodal approach. The roots of neuroblastoma plasticity and heterogeneity remain poorly understood. Here, the authors characterise the transcriptional states of neuroblastoma cells and their changes throughout development using avian embryo models, single-cell RNA-sequencing, and patient samples; they find transcriptomic changes related to early sympatho-adrenal development.

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors. In high-risk neuroblastoma cases, gains in chromosome 17q are common. Here, the authors investigate the epigenomics and transcriptomics of neuroblastoma, identifying TBX2 as a core regulatory circuitry component enhancing the reactivation of DREAM targets by MYCN/FOXM1.

Neuroblastoma is the most common extracranial solid tumor in children. A subgroup of high-risk patients is characterized by aberrations in the chromatin remodeller ATRX that is encoded by 35 exons. In contrast to other pediatric cancer where ATRX point mutations are most frequent, multi-exon deletions (MEDs) are the most frequent type of ATRX aberrations in neuroblastoma. 75% of these MEDs are predicted to produce in-frame fusion proteins, suggesting a potential gain-of-function effect compared to nonsense mutations. For neuroblastoma there are only a few patient-derived ATRX aberrant models. Therefore, we created isogenic ATRX aberrant models using CRISPR-Cas9 in several neuroblastoma cell lines and one tumoroid and performed total RNA-sequencing on these and the patient-derived models. Gene set enrichment analysis (GSEA) showed decreased expression of genes related to both ribosome biogenesis and several metabolic processes in our isogenic ATRX exon 2–10 MED model systems, the patient-derived MED models and in tumor data containing two patients with an ATRX exon 2–10 MED. In sharp contrast, these same processes showed an increased expression in our isogenic ATRX knock-out and exon 2–13 MED models. Our validations confirmed a role of ATRX in the regulation of ribosome homeostasis. The two distinct molecular expression patterns within ATRX aberrant neuroblastomas that we identified imply that there might be a need for distinct treatment regimens.

Neuroblastoma is the most common extracranial solid tumor in children. The chromatin remodeler ATRX is frequently mutated in high‐risk patients with a poor prognosis. Although many studies have reported ATRX aberrations and the associated clinical characteristics in neuroblastoma, a comprehensive overview is currently lacking. In this study, we extensively characterize the mutational spectrum of ATRX aberrations in neuroblastoma tumors reported in previous studies and present an overview of patient and tumor characteristics. We collected the data of a total of 127 neuroblastoma patients and three cell lines with ATRX aberrations originating from 20 papers. We subdivide the ATRX aberrations into nonsense, missense, and multiexon deletions (MEDs) and show that 68% of them are MEDs. Of these MEDs, 75% are predicted to be in‐frame. Furthermore, we identify a missense mutational hotspot region in the helicase domain. We also confirm that all three ATRX mutation types are more often identified in patients diagnosed at an older age, but still approximately 40% of the patients are aged 5 years or younger at diagnosis. Surprisingly, we found that 11q deletions are enriched in neuroblastomas with ATRX deletions compared to a reference cohort, but not in neuroblastomas with ATRX point mutations. Taken together, our data emphasizes a distinct ATRX mutation spectrum in neuroblastoma, which should be considered when studying molecular phenotypes and therapeutic strategies. The ATRX chromatin remodeller is mutated in up to 8.6% of high‐risk neuroblastoma patients. We show that ATRX multiexon deletions (MEDs) are almost unique to neuroblastoma compared to other pediatric cancers and that 75% of these MEDs are predicted to be in‐frame. Additionally, we found a missense mutational hotspot region in the helicase domain and show that 11q deletions, which are associated with poor survival, are strongly enriched in tumors containing ATRX MEDs, but not in tumors containing ATRX point mutations.

Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetically lethal to neuroblastoma cells with MYCN amplification and over-expression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification, and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by 3 RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53, and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetically lethal relationship between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics.