MicroRNAs (miRNAs) have emerged as fundamental regulators that silence gene expression at the post-transcriptional and translational levels. The identification of their targets is a major challenge to elucidate the regulated biological processes. The overall effect of miRNA is reflected on target mRNA expression, suggesting the design of new investigative methods based on high-throughput experimental data such as miRNA and transcriptome profiles. We propose a novel statistical measure of non-linear dependence between miRNA and mRNA expression, in order to infer miRNA-target interactions. This approach, which we name antagonism pattern detection, is based on the statistical recognition of a triangular-shaped pattern in miRNA-target expression profiles. This pattern is observed in miRNA-target expression measurements since their simultaneously elevated expression is statistically under-represented in the case of miRNA silencing effect. The proposed method enables miRNA target prediction to strongly rely on cellular context and physiological conditions reflected by expression data. The procedure has been assessed on synthetic datasets and tested on a set of real positive controls. Then it has been applied to analyze expression data from Ewing's sarcoma patients. The antagonism relationship is evaluated as a good indicator of real miRNA-target biological interaction. The predicted targets are consistently enriched for miRNA binding site motifs in their 3'UTR. Moreover, we reveal sets of predicted targets for each miRNA sharing important biological function. The procedure allows us to infer crucial miRNA regulators and their potential targets in Ewing's sarcoma disease. It can be considered as a valid statistical approach to discover new insights in the miRNA regulatory mechanisms.

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.

Rhabdomyosarcomas (RMS) in children and adolescents are heterogeneous sarcomas broadly defined by skeletal muscle features and the presence/absence of PAX3/7-FOXO1 fusion genes. MicroRNAs are small non-coding RNAs that regulate gene expression in a cell context specific manner. Sequencing analyses of microRNAs in 64 RMS revealed expression patterns separating skeletal muscle, fusion gene positive and negative RMS. Integration with parallel gene expression data assigned biological functions to 12 co-expression networks/modules that reassuringly included myogenic roles strongly correlated with microRNAs known in myogenesis and RMS development. Modules also correlated with clinical outcome and fusion status. Regulation of microRNAs by the fusion protein was demonstrated after PAX3-FOXO1 reduction, exemplified by miR-9-5p. MiR-9-5p levels correlated with poor outcome, even within fusion gene positive RMS, and were higher in metastatic versus non-metastatic disease. MiR-9-5p reduction inhibited RMS cell migration. Our findings reveal microRNAs in a regulatory framework of biological and clinical significance in RMS.

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.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by PAX3/7-FOXO1 fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human PAX3-FOXO1 -driven zebrafish models of tumorigenesis and found that PAX3-FOXO1 exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6-19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this PAX3-FOXO1 transgenic zebrafish model to study how PAX3-FOXO1 leverages early developmental pathways for oncogenesis and found that her3 is a unique target. Ectopic expression of the her3 human ortholog, HES3, inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, HES3 is overexpressed in fusion-positive versus fusion-negative tumors. Finally, HES3 overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new PAX3-FOXO1 target, her3/HES3, that contributes to impaired myogenic differentiation and has prognostic significance in human disease.

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.

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations.

Pure invasive micropapillary carcinoma (IMPC) is a special type of breast carcinoma characterised by clusters of cells presenting polarity abnormalities. The biological alterations underlying this pattern remain unknown.

The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC- 1 parental cells in nude mice generated various tumor types, such as NB, osteo/ chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.

This study investigated relationships between neuroblastomas (NBs) imaging phenotypes, tumor genomic profile and patient outcome.

The genetic cause of some familial nonsyndromic renal cell carcinomas (RCC) defined by at least two affected first-degree relatives is unknown. By combining whole-exome sequencing and tumor profiling in a family prone to cases of RCC, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC-affected relatives. Furthermore, BAP1 was found to be inactivated in RCC-affected individuals from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We then screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the recently described BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma. Among the 11 families that included individuals identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC-predisposition gene.

Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

The fusion between EWS and ETS family members is a key oncogenic event in Ewing tumors and important EWS-FLI1 target genes have been identified. However, until now, the search for EWS-FLI1 targets has been limited to promoter regions and no genome-wide comprehensive analysis of in vivo EWS-FLI1 binding sites has been undertaken. Using a ChIP-Seq approach to investigate EWS-FLI1-bound DNA sequences in two Ewing cell lines, we show that this chimeric transcription factor preferentially binds two types of sequences including consensus ETS motifs and microsatellite sequences. Most bound sites are found outside promoter regions. Microsatellites containing more than 9 GGAA repeats are very significantly enriched in EWS-FLI1 immunoprecipitates. Moreover, in reporter gene experiments, the transcription activation is highly dependent upon the number of repeats that are included in the construct. Importantly, in vivo EWS-FLI1-bound microsatellites are significantly associated with EWS-FLI1-driven gene activation. Put together, these results point out the likely contribution of microsatellite elements to long-distance transcription regulation and to oncogenesis.

The combined deletion of 1p and 19q chromosomal arms is frequent in oligodendrogliomas (OD) and has recently been shown to be mediated by an unbalanced t(1;19) translocation. Recent studies of 1p/19q co-deleted OD suggest that the NOTCH2 gene is implicated in oligodendrocyte differentiation and may be involved in this rearrangement. The objective of the present study was to analyze the NOTCH2 locus either as a chromosomal translocation locus that may be altered by the 1p/19q recurrent rearrangement or as a gene that may be inactivated by a two hit process. We performed an array-CGH analysis of 15 ODs presenting 1p/19q co-deletion using a high-density oligonucleotide microarray spanning 1p and 19q pericentromeric regions with 377 bp average probe spacing. We showed that the 1p deletion extends to the centromere of chromosome 1 and includes the entire NOTCH2 gene. No internal rearrangement of this gene was observed. This strongly suggests that the t(1;19) translocation does not lead to an abnormal NOTCH2 structure. The analysis of the entire NOTCH2 coding sequence was performed in four cases and did not reveal any mutation therefore indicating that NOTCH2 does not harbor genetic characteristics of a tumor suppressor gene. Finally, the detailed analysis of chromosome 19 pericentromeric region led to the identification of two breakpoint clusters at 19p12 and 19q11-12. Interestingly, these two regions share a large stretch of homology. Together with previous observations of similarities between chromosome 1 and 19 alphoid sequences, this suggests that the t(1;19) translocation arises from complex intra and interchromosomal rearrangements.This is the first comprehensive deletion mapping by high density oligo-array of the 1p/19q co-deletion in oligodendroglioma tumors using a methodological approach superior to others previously applied. As such this paper provides clear evidence that the NOTCH2 gene is not physically rearranged by t(1;19) translocation of oligodendroglioma tumors.

Ependymoma with RELA fusion has been defined as a novel entity of the revised World Health Organization 2016 classification of tumors of the central nervous system (CNS), characterized by fusion transcripts of the RELA gene and consequent pathological activation of the NFkB pathway. These tumors represent the majority of supratentorial ependymomas in children. The validation of diagnostic tools to identify this clinically relevant ependymoma entity is essential. Here, we have used interphase fluorescent in situ hybridization (FISH) for C11orf95 and RELA, immunohistochemistry (IHC) for p65-RelA and the recently developed DNA methylation-based classification besides conventional histopathology, and compared the precision of the methods in 40 supratentorial pediatric brain tumors diagnosed as ependymomas in the past years. Reverse transcription PCR (RT-PCR) and RNA sequencing were performed to explore discordant cases. Furthermore, we integrated imaging and clinical features as additional layers of information. The concordance between nuclear RelA expression by IHC and RELA FISH was 100%. Concordance between IHC and DNA methylation profiling, and between FISH and DNA methylation profiling was also high (96.4% and 95.2%, respectively). Thirty-four out of 40 (85%) cases were confirmed by integrated diagnoses as ependymal tumors, including 22 RELA-fused ependymomas (71% of ependymal tumors), two YAP1-fused ependymomas (6%), six non-RELA/non-YAP1 ependymomas (18%) and four ependymal/subependymal mixed tumors (12%). Ependymal/subependymal mixed tumors had an excellent clinical outcome despite the presence of histopathological signs of malignancy, suggesting that these tumors should not be diagnosed as classic ependymomas. DNA methylation profiling helped in the differential diagnosis of RELA-fused ependymomas. IHC and FISH, which are available in the majority of pathology laboratories, are valuable tools to identify RELA-fused ependymomas.

Pilocytic astrocytoma (PA) is the most common pediatric glioma, arising from a single driver MAPK pathway alteration. Classified as a grade I tumor according to the 2016 WHO classification, prognosis is excellent with a 10-year survival rate > 95% after surgery. However, rare cases present with anaplastic features, including an unexpected high mitotic/proliferative index, thus posing a diagnostic and therapeutic challenge. Based on small histomolecular series and case reports, such tumors arising at the time of diagnosis or recurrence have been designated by many names including pilocytic astrocytoma with anaplastic features (PAAF). Recent DNA methylation-profiling studies performed mainly on adult cases have revealed that PAAF exhibit a specific methylation signature, thus constituting a distinct methylation class from typical PA [methylation class anaplastic astrocytoma with piloid features-(MC-AAP)]. However, the diagnostic and prognostic significance of MC-AAP remains to be determined in children. We performed an integrative work on the largest pediatric cohort of PAAF, defined according to strict criteria: morphology compatible with the diagnosis of PA, with or without necrosis, ≥ 4 mitoses for 2.3 mm2, and MAPK pathway alteration. We subjected 31 tumors to clinical, imaging, morphological and molecular analyses, including DNA methylation profiling. We identified only one tumor belonging to the MC-AAP (3%), the others exhibiting a methylation profile typical for PA (77%), IDH-wild-type glioblastoma (7%), and diffuse leptomeningeal glioneuronal tumor (3%), while three cases (10%) did not match to a known DNA methylation class. No significant outcome differences were observed between PAAF with necrosis versus no necrosis (p = 0.07), or with 4-6 mitoses versus 7 or more mitoses (p = 0.857). Our findings argue that the diagnostic histomolecular criteria established for anaplasia in adult PA are not of diagnostic or prognostic value in a pediatric setting. Further extensive and comprehensive integrative studies are necessary to accurately define this exceptional entity in children.

Ewing sarcoma (EwS) is an aggressive primary bone cancer in children and young adults characterized by oncogenic fusions between genes encoding FET-RNA-binding proteins and ETS transcription factors, the most frequent fusion being EWSR1-FLI1. We show that EGR2, an Ewing-susceptibility gene and an essential direct target of EWSR1-FLI1, directly regulates the transcription of genes encoding key enzymes of the mevalonate (MVA) pathway. Consequently, Ewing sarcoma is one of the tumors that expresses the highest levels of mevalonate pathway genes. Moreover, genome-wide screens indicate that MVA pathway genes constitute major dependencies of Ewing cells. Accordingly, the statin inhibitors of HMG-CoA-reductase, a rate-limiting enzyme of the MVA pathway, demonstrate cytotoxicity in EwS. Statins induce increased ROS and lipid peroxidation levels, as well as decreased membrane localization of prenylated proteins, such as small GTP proteins. These metabolic effects lead to an alteration in the dynamics of S-phase progression and to apoptosis. Statin-induced effects can be rescued by downstream products of the MVA pathway. Finally, we further show that statins impair tumor growth in different Ewing PDX models. Altogether, the data show that statins, which are off-patent, well-tolerated, and inexpensive compounds, should be strongly considered in the therapeutic arsenal against this deadly childhood disease.

Medulloblastoma (MB) is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFβ/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. We showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation. Beside genetic alterations, we demonstrated that an ActivinB autocrine stimulation is responsible for pathway activation in a subset of Group 3 MB characterized by high PMEPA1 levels. Importantly, Galunisertib, a kinase inhibitor of the cognate receptors currently tested in clinical trials for Glioblastoma patients, showed efficacy on orthotopically grafted MB-PDX. Our data demonstrate that the TGFβ/Activin pathway is active in a subset of Group 3 MB and can be therapeutically targeted.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc - transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659.