A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies.

Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine ß-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.

MYCN and ALKF1174L are sufficient to drive neuroblastoma development from neural crest progenitor cells.

Neuroblastoma is an embryonal tumor with a heterogeneous clinical course. The tumor is presumed to be derived from the neural crest, but the cells of origin remain to be determined. To date, few recurrent genetic changes contributing to neuroblastoma formation, such as amplification of the MYCN oncogene and activating mutations of the ALK oncogene, have been identified. The possibility to model neuroblastoma in mice allows investigation of the cell of origin hypothesis in further detail. Here we present the evidence that murine neural crest progenitor cells can give rise to neuroblastoma upon transformation with MYCN or ALK(F1174L). For this purpose we used JoMa1, a multipotent neural crest progenitor cell line, which is kept in a viable and undifferentiated state by a tamoxifen-activated c-Myc transgene (c-MycER(T)). Expression of MYCN or ALK(F1174L), one of the oncogenic ALK variants identified in primary neuroblastomas, enabled these cells to grow independently of c-MycER(T) activity in vitro and caused formation of neuroblastoma-like tumors in vivo in contrast to parental JoMa1 cells and JoMa1 cells-expressing TrkA or GFP. Tumorigenicity was enhanced upon serial transplantation of tumor-derived cells, and tumor cells remained susceptible to the MYC-inhibitor, NBT-272, indicating that cell growth depended on functional MYCN. Our findings support neural crest progenitor cells as the precursor cells of neuroblastoma, and indicate that neuroblastomas arise as their malignant progeny.

Exon-level expression analyses identify MYCN and NTRK1 as major determinants of alternative exon usage and robustly predict primary neuroblastoma outcome.

BACKGROUND: Using mRNA expression-derived signatures as predictors of individual patient outcome has been a goal ever since the introduction of microarrays. Here, we addressed whether analyses of tumour mRNA at the exon level can improve on the predictive power and classification accuracy of gene-based expression profiles using neuroblastoma as a model. METHODS: In a patient cohort comprising 113 primary neuroblastoma specimens expression profiling using exon-level analyses was performed to define predictive signatures using various machine-learning techniques. Alternative transcript use was calculated from relative exon expression. Validation of alternative transcripts was achieved using qPCR- and cell-based approaches. RESULTS: Both predictors derived from the gene or the exon levels resulted in prediction accuracies >80% for both event-free and overall survival and proved as independent prognostic markers in multivariate analyses. Alternative transcript use was most prominently linked to the amplification status of the MYCN oncogene, expression of the TrkA/NTRK1 neurotrophin receptor and survival. CONCLUSION: As exon level-based prediction yields comparable, but not significantly better, prediction accuracy than gene expression-based predictors, gene-based assays seem to be sufficiently precise for predicting outcome of neuroblastoma patients. However, exon-level analyses provide added knowledge by identifying alternative transcript use, which should deepen the understanding of neuroblastoma biology.

An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours.

Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.

MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c-MYC-activated tumors.

Increased activity of MYC protein-family members is a common feature in many cancers. Using neuroblastoma as a tumor model, we established a microRNA (miRNA) signature for activated MYCN/c-MYC signaling in two independent primary neuroblastoma tumor cohorts and provide evidence that c-MYC and MYCN have overlapping functions. On the basis of an integrated approach including miRNA and messenger RNA (mRNA) gene expression data we show that miRNA activation contributes to widespread mRNA repression, both in c-MYC- and MYCN-activated tumors. c-MYC/MYCN-induced miRNA activation was shown to be dependent on c-MYC/MYCN promoter binding as evidenced by chromatin immunoprecipitation. Finally, we show that pathways, repressed through c-MYC/MYCN miRNA activation, are highly correlated to tumor aggressiveness and are conserved across different tumor entities suggesting that c-MYC/MYCN activate a core set of miRNAs for cooperative repression of common transcriptional programs related to disease aggressiveness. Our results uncover a widespread correlation between miRNA activation and c-MYC/MYCN-mediated coding gene expression modulation and further substantiate the overlapping functions of c-MYC and MYCN in the process of tumorigenesis.

Copy number alterations and copy number variation in cancer: close encounters of the bad kind.

Recent studies have unveiled copy number variants (CNVs) as an important source of genetic variation. Many of these CNVs contain coding sequences, which have been shown to be dosage sensitive. Evidence is accumulating that certain CNVs have impact on susceptibility to human diseases such as HIV infection and autoimmune diseases, as well as on adaptability to environmental conditions or nutrition. The possible role and impact of CNVs on cancer development and progression is only now emerging. In this review we look into the role of CNVs and their associated genomic structural features in relation to the formation of chromosome alterations in cancer cells and evolutionary genomic plasticity, as well as the de novo occurrence of known or putative CNVs as somatic events during oncogenesis. The role of germline CNVs in cancer predisposition is still largely unexplored. A number of observations seem to warrant the importance of further studies to elucidate the impact of these variants in the early steps of carcinogenesis.

[New insights into the genetic basis of neuroblastoma]. / Nieuwe inzichten in de genetische basis van neuroblastoom.

Neuroblastoma (NB) is, next to acute lymphoblastic leukaemia, brain tumours and lymphoma the most frequent paediatric tumour (8-10%). Our research group aims to contribute to the unravelling of the genetic basis of NB. Insight into the genes and signalling pathways involved in tumour formation and development can represent an essential step towards the development of more efficient molecular targeted therapies. A first part of our research work was devoted to the analysis of genomic alterations in NB. By means of a new highly sensitive method for detecting gains and losses of chromosomal segments, we recognised three major prognostic relevant genomic subtypes of NB. In addition smaller subgroups with deviating genomic patterns were detected. In addition, this work yielded important information regarding delineation of critical regions of gain and loss in NB which should facilitate further selection of candidate oncogenes or tumour suppressor genes. A second important part of our work focussed on the gene expression profiling of NB precursor cells. We were able as the first to isolate these cells and determine their transcriptome, a finding of fundamental importance for future expression studies in NB. Another study focussed on the identification of MYCN transcriptional target genes. Gene expression analyses of model systems developed in our lab and of a large panel of cell lines and tumours allowed us to subtract a list of candidate genes which are now under further study. Finally, we initiated research towards the understanding of the role of methylation in NB oncogenesis. From this, we were able to create a list of potentially relevant methylated genes in NB. From the above it is clear that our team has made important contributions to the understanding of the complex biology and clinical behaviour of NB. Also, a broad technically innovative research platform has been developed which will allow us to dissect NB genetics with greater speed and accuracy.

Genome wide measurement of DNA copy number changes in neuroblastoma: dissecting amplicons and mapping losses, gains and breakpoints.

In the past few years high throughput methods for assessment of DNA copy number alterations have witnessed rapid progress. Both 'in house' developed BAC, cDNA, oligonucleotide and commercial arrays are now available and widely applied in the study of the human genome, particularly in the context of disease. Cancer cells are known to exhibit DNA losses, gains and amplifications affecting tumor suppressor genes and proto-oncogenes. Moreover, these patterns of genomic imbalances may be associated with particular tumor types or subtypes and may have prognostic value. Here we summarize recent array CGH findings in neuroblastoma, a pediatric tumor of the sympathetic nervous system. A total of 176 primary tumors and 53 cell lines have been analyzed on different platforms. Through these studies the genomic content and boundaries of deletions, gains and amplifications were characterized with unprecedented accuracy. Furthermore, in conjunction with cytogenetic findings, array CGH allows the mapping of breakpoints of unbalanced translocations at a very high resolution.