No abstract

Neuroblastoma and other pediatric tumors show a paucity of gene mutations, which has sparked an interest in their epigenetic regulation. Several tumor types include phenotypically divergent cells, resembling cells from different lineage development stages. It has been proposed that super-enhancer-associated transcription factor (TF) networks underlie lineage identity, but the role of these enhancers in intratumoral heterogeneity is unknown. Here we show that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal cells and committed adrenergic cells can interconvert and resemble cells from different lineage differentiation stages. ChIP-seq analysis of isogenic pairs of mesenchymal and adrenergic cells identified a distinct super-enhancer landscape and super-enhancer-associated TF network for each cell type. Expression of the mesenchymal TF PRRX1 could reprogram the super-enhancer and mRNA landscapes of adrenergic cells toward a mesenchymal state. Mesenchymal cells were more chemoresistant in vitro and were enriched in post-therapy and relapse tumors. Two super-enhancer-associated TF networks, which probably mediate lineage control in normal development, thus dominate epigenetic control of neuroblastoma and shape intratumoral heterogeneity.

Whole-genome sequencing detected structural rearrangements of TERT in 17 of 75 high-stage neuroblastomas, with five cases resulting from chromothripsis. Rearrangements were associated with increased TERT expression and targeted regions immediately up- and downstream of TERT, positioning a super-enhancer close to the breakpoints in seven cases. TERT rearrangements (23%), ATRX deletions (11%) and MYCN amplifications (37%) identify three almost non-overlapping groups of high-stage neuroblastoma, each associated with very poor prognosis.

The myc oncogenes are frequently activated in human tumors, but there is no comprehensive insight into the target genes and downstream cellular pathways of these transcription factors. We applied serial analysis of gene expression (SAGE) to identify targets of N‐myc in neuroblastomas. Analysis of 42 000 mRNA transcript tags in SAGE libraries of N‐myc‐ transfected and control neuroblastoma cells revealed 114 up‐regulated genes. The majority of these genes have a role in ribosome assembly and activity. Northern blot analysis confirmed up‐regulation of all tested transcripts. Induction was complete within 4 h after N‐myc expression. The large majority of the ribosomal proteins were induced, as well as genes controlling rRNA maturation. Cellular rRNA content was 45% induced. SAGE libraries and northern blot analysis confirmed up‐regulation of many of these genes in N‐myc‐amplified neuroblastomas. As N‐myc can functionally replace c‐myc, we analyzed whether N‐myc targets were induced by c‐myc as well. Approximately 40% of these N‐myc targets were up‐regulated in a c‐myc‐transfected melanoma cell line. These data suggest that myc genes function as major regulators of the protein synthesis machinery.

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B-let-7-MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

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. N euroblastoma is a pediatric solid tumor derived from the sympathetic nervous system. The prognosis is highly variable and is associated with parameters such as age at diagnosis, dissemination at time of diagnosis, tumor stage, and grade of differentiation of the primary tumor (1). Neuroblastoma stages 1 and 2 have a very good prognosis, but survival in stage 4 neuroblastoma is below 30%. Amplification of MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is associated with poor outcome. It occurs in about 20% of the tumors but is confined to high-stage neuroblastoma.Together with MYC and MYCL, MYCN belongs to the MYC transcription factor family, whose role in cancer has been studied extensively. A series of investigators have manipulated MYCN expression in neuroblastoma cell lines by overexpression or silencing. High expression of MYCN is associated with fast proliferation and induction of cell cycle genes [(2-5) and reviewed by Bell (5)]. Although cell cycle genes were identified, the actual number of MYC-regulated genes is a small minority compared with all other genes claimed to be regulated in such experiments. In fact, thousands of candidate target genes of MYC and/or MYCN currently are known, which has complicated pinpointing of the relevant set of genes regulated by MYC genes and responsible for the aggressive phenotype.In a very early study, MYCN protein expression was found to be a poor prognostic factor (6). Recent experiments in cell lines showed that MYCN protein stability is decreased after phosphorylation by glycogen synthas...

We have investigated the peripheral myelin protein gene, PMP-22, in a family with Charcot-Marie-Tooth disease type 1A (CMT1A). The DNA duplication commonly found in CMT1A was absent in this family, but strong linkage existed between the disease and the CMT1A marker VAW409R3 on chromosome 17p11.2. We found a point mutation in PMP-22 which was completely linked with the disease. The mutation, a proline for leucine substitution in the first putative transmembrane domain, is identical to that recently found in the Trembler-J mouse. The presence of this PMP-22 defect in this CMT1A family and the location of PMP-22 within the DNA duplication associated with CMT1A suggest that both structural alteration and overexpression of PMP-22 may lead to the disease.

Transition between differentiation states in development occurs swift but the mechanisms leading to epigenetic and transcriptional reprogramming are poorly understood. The pediatric cancer neuroblastoma includes adrenergic (ADRN) and mesenchymal (MES) tumor cell types, which differ in phenotype, super-enhancers (SEs) and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we unravel how a NOTCH3 intracellular domain reprogrammed the ADRN transcriptional landscape towards a MES state. A transcriptional feed-forward circuitry of NOTCH-family transcription factors amplifies the NOTCH signaling levels, explaining the swift transition between two semi-stable cellular states. This transition induces genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. Once established, the NOTCH feed-forward loop maintains the induced MES state. In vivo reprogramming of ADRN cells shows that MES and ADRN cells are equally oncogenic. Our results elucidate a swift transdifferentiation between two semi-stable epigenetic cellular states.