Chemotherapies active in preclinical studies frequently fail in the clinic due to lack of efficacy, which limits progress for rare cancers since only small numbers of patients are available for clinical trials. Thus, a preclinical drug development pipeline was developed to prioritize potentially active regimens for pediatric brain tumors spanning from in vitro drug screening, through intracranial and intra-tumoral pharmacokinetics to in vivo efficacy studies. Here, as an example of the pipeline, data are presented for the combination of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in three pediatric brain tumor models. The in vitro activity of nine novel therapies was tested against tumor spheres derived from faithful mouse models of Group 3 medulloblastoma, ependymoma, and choroid plexus carcinoma. Agents with the greatest in vitro potency were then subjected to a comprehensive series of in vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies culminating in preclinical efficacy trials in mice harboring brain tumors. The nucleoside analog 5-fluoro-2'-deoxycytidine (FdCyd) markedly reduced the proliferation in vitro of all three brain tumor cell types at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in brain tumors necessary to inhibit tumor cell proliferation, but no tumor displayed a significant in vivo therapeutic response. Despite promising in vitro activity and in vivo PK properties, FdCyd is unlikely to be an effective treatment of pediatric brain tumors, and therefore was deprioritized for the clinic. Our comprehensive and integrated preclinical drug development pipeline should reduce the attrition of drugs in clinical trials.

TET-family dioxygenases oxidize 5-methylcytosine (5mC) in DNA, and exert tumour suppressor activity in many types of cancers. Even in the absence of TET coding region mutations, TET loss-of-function is strongly associated with cancer. Here we show that acute elimination of TET function induces the rapid development of an aggressive, fully-penetrant and cell-autonomous myeloid leukaemia in mice, pointing to a causative role for TET loss-of-function in this myeloid malignancy. Phenotypic and transcriptional profiling shows aberrant differentiation of haematopoietic stem/progenitor cells, impaired erythroid and lymphoid differentiation and strong skewing to the myeloid lineage, with only a mild relation to changes in DNA modification. We also observe progressive accumulation of phospho-H2AX and strong impairment of DNA damage repair pathways, suggesting a key role for TET proteins in maintaining genome integrity.

Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail.Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG.Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis (11.1 and 10.8 months median overall survival, respectively). Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization.These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones.H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implication for the design of future trials in these specific types of neoplasms.

The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the (epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.

Gains and losses in DNA methylation are prominent features of mammalian cell types. To gain insight into the mechanisms that promote shifts in DNA methylation and contribute to changes in cell fate, including malignant transformation, we performed genome-wide mapping of 5-methylcytosine and 5-hydroxymethylcytosine in purified mouse hematopoietic stem cells. We discovered extended regions of low methylation (canyons) that span conserved domains frequently containing transcription factors and are distinct from CpG islands and shores. About half of the genes in these methylation canyons are coated with repressive histone marks, whereas the remainder are covered by activating histone marks and are highly expressed in hematopoietic stem cells (HSCs). Canyon borders are demarked by 5-hydroxymethylcytosine and become eroded in the absence of DNA methyltransferase 3a (Dnmt3a). Genes dysregulated in human leukemias are enriched for canyon-associated genes. The new epigenetic landscape we describe may provide a mechanism for the regulation of hematopoiesis and may contribute to leukemia development.

Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells.

DNA enrichment followed by sequencing is a versatile tool in molecular biology, with a wide variety of applications including genome-wide analysis of epigenetic marks and mechanisms. A common requirement of these diverse applications is a comparison of read coverage between experimental conditions. The amount of samples generated for such comparisons ranges from few replicates to hundreds of samples per condition for epigenome-wide association studies. Consequently, there is an urgent need for software that allows for fast and simple processing and comparison of sequencing data derived from enriched DNA.

Recurrent medulloblastoma is a therapeutic challenge because it is almost always fatal. Studies have confirmed that medulloblastoma consists of at least four distinct subgroups. We sought to delineate subgroup-specific differences in medulloblastoma recurrence patterns.

Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in ∼50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a global reduction of the repressive histone mark H3K27me3. We demonstrate that this is caused by aberrant recruitment of the PRC2 complex to K27M mutant H3.3 and enzymatic inhibition of the H3K27me3-establishing methyltransferase EZH2. By performing chromatin immunoprecipitation followed by next-generation sequencing and whole-genome bisulfite sequencing in primary pHGGs, we show that reduced H3K27me3 levels and DNA hypomethylation act in concert to activate gene expression in K27M mutant pHGGs.

Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES) cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas "gene regulatory hotspots" which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints.

Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.

A hallmark of pediatric low-grade glioma (pLGG) is aberrant signaling of the mitogen activated protein kinase (MAPK) pathway. Hence, inhibition of MAPK signaling using small molecule inhibitors such as MEK inhibitors (MEKi) may be a promising strategy.

Spinal ependymal tumors form a histologically and molecularly heterogeneous group of tumors with generally good prognosis. However, their treatment can be challenging if infiltration of the spinal cord or dissemination throughout the central nervous system (CNS) occurs and, in these cases, clinical outcome remains poor. Here, we describe a new and relatively rare subgroup of spinal ependymal tumors identified using DNA methylation profiling that is distinct from other molecular subgroups of ependymoma. Copy number variation plots derived from DNA methylation arrays showed MYCN amplification as a characteristic genetic alteration in all cases of our cohort (n = 13), which was subsequently validated using fluorescence in situ hybridization. The histological diagnosis was anaplastic ependymoma (WHO Grade III) in ten cases and classic ependymoma (WHO Grade II) in three cases. Histological re-evaluation in five primary tumors and seven relapses showed characteristic histological features of ependymoma, namely pseudorosettes, GFAP- and EMA positivity. Electron microscopy revealed cilia, complex intercellular junctions and intermediate filaments in a representative sample. Taking these findings into account, we suggest to designate this molecular subgroup spinal ependymoma with MYCN amplification, SP-EPN-MYCN. SP-EPN-MYCN tumors showed distinct growth patterns with intradural, extramedullary localization mostly within the thoracic and cervical spine, diffuse leptomeningeal spread throughout the whole CNS and infiltrative invasion of the spinal cord. Dissemination was observed in 100% of cases. Despite high-intensity treatment, SP-EPN-MYCN showed significantly worse median progression free survival (PFS) (17 months) and median overall survival (OS) (87 months) than all other previously described molecular spinal ependymoma subgroups. OS and PFS were similar to supratentorial ependymoma with RELA-fusion (ST-EPN-RELA) and posterior fossa ependymoma A (PF-EPN-A), further highlighting the aggressiveness of this distinct new subgroup. We, therefore, propose to establish SP-EPN-MYCN as a new molecular subgroup in ependymoma and advocate for testing newly diagnosed spinal ependymal tumors for MYCN amplification.

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.

Molecular groups of medulloblastoma (MB) are well established. Novel risk stratification parameters include Group 3/4 (non-WNT/non-SHH) methylation subgroups I-VIII or whole-chromosomal aberration (WCA) phenotypes. This study investigates the integration of clinical and molecular parameters to improve risk stratification of non-WNT/non-SHH MB. Non-WNT/non-SHH MB from the HIT2000 study and the HIT-MED registries were selected based on availability of DNA-methylation profiling data. MYC or MYCN amplification and WCA of chromosomes 7, 8, and 11 were inferred from methylation array-based copy number profiles. In total, 403 non-WNT/non-SHH MB were identified, 346/403 (86%) had a methylation class family Group 3/4 methylation score (classifier v11b6) ≥ 0.9, and 294/346 (73%) were included in the risk stratification modeling based on Group 3 or 4 score (v11b6) ≥ 0.8 and subgroup I-VIII score (mb_g34) ≥ 0.8. Group 3 MB (5y-PFS, survival estimation ± standard deviation: 41.4 ± 4.6%; 5y-OS: 48.8 ± 5.0%) showed poorer survival compared to Group 4 (5y-PFS: 68.2 ± 3.7%; 5y-OS: 84.8 ± 2.8%). Subgroups II (5y-PFS: 27.6 ± 8.2%) and III (5y-PFS: 37.5 ± 7.9%) showed the poorest and subgroup VI (5y-PFS: 76.6 ± 7.9%), VII (5y-PFS: 75.9 ± 7.2%), and VIII (5y-PFS: 66.6 ± 5.8%) the best survival. Multivariate analysis revealed subgroup in combination with WCA phenotype to best predict risk of progression and death. The integration of clinical (age, M and R status) and molecular (MYC/N, subgroup, WCA phenotype) variables identified a low-risk stratum with a 5y-PFS of 94 ± 5.7 and a very high-risk stratum with a 5y-PFS of 29 ± 6.1%. Validation in an international MB cohort confirmed the combined stratification scheme with 82.1 ± 6.0% 5y-PFS in the low and 47.5 ± 4.1% in very high-risk groups, and outperformed the clinical model. These newly identified clinico-molecular low-risk and very high-risk strata, accounting for 6%, and 21% of non-WNT/non-SHH MB patients, respectively, may improve future treatment stratification.

Choroid plexus carcinoma (CPC) is a rare and aggressive tumor of infancy without a clear treatment strategy. This study describes the outcomes of children with CPC treated on the multi-institutional phase 2 SJYC07 trial and reports on the significance of clinical and molecular characteristics.