Mesenchymal-Type Neuroblastoma Cells Escape ALK Inhibitors.

Cancer therapy frequently fails due to the emergence of resistance. Many tumors include phenotypically immature tumor cells, which have been implicated in therapy resistance. Neuroblastoma cells can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) state. They differ in epigenetic landscape and transcription factors, and MES cells are more resistant to chemotherapy. Here we analyzed the response of MES cells to targeted drugs. Activating anaplastic lymphoma kinase (ALK) mutations are frequently found in neuroblastoma and ALK inhibitors (ALKi) are in clinical trials. ALKi treatment of ADRN neuroblastoma cells with a tumor-driving ALK mutation induced cell death. Conversely, MES cells did not express either mutant or wild-type ALK and were resistant to ALKi, and MES cells formed tumors that progressed under ALKi therapy. In assessing the role of MES cells in relapse development, TRAIL was identified to specifically induce apoptosis in MES cells and to suppress MES tumor growth. Addition of TRAIL to ALKi treatment of neuroblastoma xenografts delayed relapses in a subset of the animals, suggesting a role for MES cells in relapse formation. While ADRN cells resembled normal embryonal neuroblasts, MES cells resembled immature precursor cells, which also lacked ALK expression. Resistance to targeted drugs can therefore be an intrinsic property of immature cancer cells based on their resemblance to developmental precursors. SIGNIFICANCE: In neuroblastoma, mesenchymal tumor cells lack expression of the tumor-driving ALK oncogene and are resistant to ALKi, but dual treatment with ALKi and mesenchymal cell-targeting TRAIL delays tumor relapse.

A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma.

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.

Neuroblastoma is composed of two super-enhancer-associated differentiation states.

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.

TERT rearrangements are frequent in neuroblastoma and identify aggressive tumors.

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.

OTX2 directly activates cell cycle genes and inhibits differentiation in medulloblastoma cells.

The transcription factor OTX2 has been implicated as an oncogene in medulloblastoma, which is the most common malignant brain tumor in children. It is highly expressed in most medulloblastomas and amplified in a subset of them. To study the role OTX2 has in medulloblastoma we investigated the downstream pathway of OTX2. We generated D425 medulloblastoma cells in which endogenous OTX2 can be silenced by inducible shRNA. Silencing of OTX2 strongly inhibited cell proliferation and resulted in a neuronal-like differentiation. Expression profiling of time courses after silencing showed a progressive change in gene expression for many cellular processes. Downregulated genes were highly enriched for cell cycle and visual perception genes, while upregulated genes were enriched for genes involved in development and differentiation. This shift is reminiscent of expression changes described during normal cerebellum development where proliferating granule progenitor cells have high OTX2 expression, which diminishes when these cells exit the cell cycle and start to differentiate. ChIP-on-chip analyses of OTX2 in D425 cells identified cell cycle and perception genes as direct OTX2 targets, while regulation of most differentiation genes appeared to be indirect. The expression of many directly regulated genes correlated to OTX2 expression in primary tumors, suggesting the in vivo relevance of these genes and their potential as targets for therapeutic intervention. These analyses provide more insight in the molecular network of OTX2, demonstrating that OTX2 is essential in medulloblastoma and directly drives proliferation by regulation of cell cycle genes.

Joint binding of OTX2 and MYC in promotor regions is associated with high gene expression in medulloblastoma.

Both OTX2 and MYC are important oncogenes in medulloblastoma, the most common malignant brain tumor in childhood. Much is known about MYC binding to promoter regions, but OTX2 binding is hardly investigated. We used ChIP-on-chip data to analyze the binding patterns of both transcription factors in D425 medulloblastoma cells. When combining the data for all promoter regions in the genome, OTX2 binding showed a remarkable bi-modal distribution pattern with peaks around -250 bp upstream and +650 bp downstream of the transcription start sites (TSSs). Indeed, 40.2% of all OTX2-bound TSSs had more than one significant OTX2-binding peak. This OTX2-binding pattern was very different from the TSS-centered single peak binding pattern observed for MYC and other known transcription factors. However, in individual promoter regions, OTX2 and MYC have a strong tendency to bind in proximity of each other. OTX2-binding sequences are depleted near TSSs in the genome, providing an explanation for the observed bi-modal distribution of OTX2 binding. This contrasts to the enrichment of E-box sequences at TSSs. Both OTX2 and MYC binding independently correlated with higher gene expression. Interestingly, genes of promoter regions with multiple OTX2 binding as well as MYC binding showed the highest expression levels in D425 cells and in primary medulloblastomas. Genes within this class of promoter regions were enriched for medulloblastoma and stem cell specific genes. Our data suggest an important functional interaction between OTX2 and MYC in regulating gene expression in medulloblastoma.

Regulation of cell cycle genes and induction of senescence by overexpression of OTX2 in medulloblastoma cell lines.

The transcription factor orthodenticle homeobox 2 (OTX2) has been implicated in the pathogenesis of medulloblastoma, as it is often highly expressed and sometimes amplified in these tumors. Little is known of the downstream pathways regulated by OTX2. We therefore generated MED8A and DAOY medulloblastoma cell lines with doxycycline-inducible OTX2 expression. In both cell lines, OTX2 inhibited proliferation and induced a senescence-like phenotype with senescence-associated ß-galactosidase activity. Expression profiles of time series after OTX2 induction in MED8A showed early upregulation of cell cycle genes related to the G(2)-M phase, such as AURKA, CDC25C, and CCNG2. Paradoxically, G(1)-S phase genes such as MYC, CDK4, CDK6, CCND1, and CCND2 were strongly downregulated, in line with the observed G(1) arrest. ChIP-on-chip analyses of OTX2 binding to promoter regions in MED8A and DAOY showed a strong enrichment for binding to the G(2)-M genes, suggesting a direct activation. Their mRNA expression correlated with OTX2 expression in primary tumors, underscoring the in vivo relevance of this regulation. OTX2 induction activated the P53 pathway in MED8A, but not in DAOY, which carries a mutated P53 gene. In DAOY cells, senescence-associated secretory factors, such as interleukin-6 and insulin-like growth factor binding protein 7, were strongly upregulated after OTX2 induction. We hypothesize that the imbalance in cell cycle stimulation by OTX2 leads to cellular senescence either by activating the P53 pathway or through the induction of secretory factors. Our data indicate that OTX2 directly induces a series of cell cycle genes but requires cooperating genes for an oncogenic acceleration of the cell cycle.

Integrated genomics identifies five medulloblastoma subtypes with distinct genetic profiles, pathway signatures and clinicopathological features.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements in cure rates, prediction of disease outcome remains a major challenge and survivors suffer from serious therapy-related side-effects. Recent data showed that patients with WNT-activated tumors have a favorable prognosis, suggesting that these patients could be treated less intensively, thereby reducing the side-effects. This illustrates the potential benefits of a robust classification of medulloblastoma patients and a detailed knowledge of associated biological mechanisms. METHODS AND FINDINGS: To get a better insight into the molecular biology of medulloblastoma we established mRNA expression profiles of 62 medulloblastomas and analyzed 52 of them also by comparative genomic hybridization (CGH) arrays. Five molecular subtypes were identified, characterized by WNT signaling (A; 9 cases), SHH signaling (B; 15 cases), expression of neuronal differentiation genes (C and D; 16 and 11 cases, respectively) or photoreceptor genes (D and E; both 11 cases). Mutations in beta-catenin were identified in all 9 type A tumors, but not in any other tumor. PTCH1 mutations were exclusively identified in type B tumors. CGH analysis identified several fully or partly subtype-specific chromosomal aberrations. Monosomy of chromosome 6 occurred only in type A tumors, loss of 9q mostly occurred in type B tumors, whereas chromosome 17 aberrations, most common in medulloblastoma, were strongly associated with type C or D tumors. Loss of the inactivated X-chromosome was highly specific for female cases of type C, D and E tumors. Gene expression levels faithfully reflected the chromosomal copy number changes. Clinicopathological features significantly different between the 5 subtypes included metastatic disease and age at diagnosis and histology. Metastatic disease at diagnosis was significantly associated with subtypes C and D and most strongly with subtype E. Patients below 3 yrs of age had type B, D, or E tumors. Type B included most desmoplastic cases. We validated and confirmed the molecular subtypes and their associated clinicopathological features with expression data from a second independent series of 46 medulloblastomas. CONCLUSIONS: The new medulloblastoma classification presented in this study will greatly enhance the understanding of this heterogeneous disease. It will enable a better selection and evaluation of patients in clinical trials, and it will support the development of new molecular targeted therapies. Ultimately, our results may lead to more individualized therapies with improved cure rates and a better quality of life.