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.
Personalized cancer treatments using combinations of drugs with a synergistic effect is attractive but proves to be highly challenging. Here we present an approach to uncover the efficacy of drug ...combinations based on the analysis of mono-drug effects. For this we used dose-response data from pharmacogenomic encyclopedias and represent these as a drug atlas. The drug atlas represents the relations between drug effects and allows to identify independent processes for which the tumor might be particularly vulnerable when attacked by two drugs. Our approach enables the prediction of combination-therapy which can be linked to tumor-driving mutations. By using this strategy, we can uncover potential effective drug combinations on a pan-cancer scale. Predicted synergies are provided and have been validated in glioblastoma, breast cancer, melanoma and leukemia mouse-models, resulting in therapeutic synergy in 75% of the tested models. This indicates that we can accurately predict effective drug combinations with translational value.
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.
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IJS, NUK, SBMB, UL, UM, UPUK
Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent ‘+4’ cells. Here, we combine transcriptomics with proteomics to define ...a definitive molecular signature for Lgr5+ CBC cells. Transcriptional profiling of FACS‐sorted Lgr5+ stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell‐enriched genes was defined. Spatial expression patterns were further characterized by mRNA in‐situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5+stem cells. Lineage tracing using a newly established knock‐in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find—and confirm by independent approaches—that the proposed quiescent/‘+4’ stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells.
Transcriptome and proteome analyses of Lgr5‐positive intestinal cells define the signature of bona fide intestinal stem cells (ISCs) population. These results offer further insight into the nature of ISCs and will instruct further research on this therapeutically highly relevant topic.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Blastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this ...subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.
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•SIX1/2 mutations in blastemal type Wilms tumors induce a proliferation signature•DROSHA/DGCR8 microprocessor mutations lead to a broad decrease in miRNA processing•TP53 mutations are associated with aneuploidy, chromothripsis, and high lethality•IGF2 and MYCN/FBXW7 alterations are frequent in blastemal type tumors
Blastemal type Wilms tumors are associated with poor prognosis. Wegert et al. identify recurrent mutations in SIX1 and SIX2 that correlate with high proliferation and in DROSHA and DGCR8 that affect miRNA biogenesis, as well as a high frequency of IGF2 overexpression in these tumors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with a highly variable prognosis. Activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway in neuroblastoma is ...correlated with poor patient prognosis, but the precise downstream effectors mediating this effect have not been determined. Here we identify the forkhead transcription factor FOXO3a as a key target of the PI3K/AKT pathway in neuroblastoma. FOXO3a expression was elevated in low-stage neuroblastoma tumors and normal embryonal neuroblasts, but reduced in late-stage neuroblastoma. Inactivation of FOXO3a by AKT was essential for neuroblastoma cell survival. Treatment of neuroblastoma cells with the dual PI3K/mTOR inhibitor PI-103 activated FOXO3a and triggered apoptosis. This effect was rescued by FOXO3a silencing. Conversely, apoptosis induced by PI-103 or the AKT inhibitor MK-2206 was potentiated by FOXO3a overexpression. Furthermore, levels of total or phosphorylated FOXO3a correlated closely with apoptotic sensitivity to MK-2206. In clinical specimens, there was an inverse relationship between gene expression signatures regulated by PI3K signaling and FOXO3a transcriptional activity. Moreover, high PI3K activity and low FOXO3a activity were each associated with an extremely poor prognosis. Our work indicates that expression of FOXO3a and its targets offer useful prognostic markers as well as biomarkers for PI3K/AKT inhibitor efficacy in neuroblastoma.
Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. ...Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA-mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene-specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy.
Neuroblastoma is characterized by substantial clinical heterogeneity. Despite intensive treatment, the survival rates of high-risk neuroblastoma patients are still disappointingly low. Somatic ...chromosomal copy number aberrations have been shown to be associated with patient outcome, particularly in low- and intermediate-risk neuroblastoma patients. To improve outcome prediction in high-risk neuroblastoma, we aimed to design a prognostic classification method based on copy number aberrations.
In an international collaboration, normalized high-resolution DNA copy number data (arrayCGH and SNP arrays) from 556 high-risk neuroblastomas obtained at diagnosis were collected from nine collaborative groups and segmented using the same method. We applied logistic and Cox proportional hazard regression to identify genomic aberrations associated with poor outcome.
In this study, we identified two types of copy number aberrations that are associated with extremely poor outcome. Distal 6q losses were detected in 5.9% of patients and were associated with a 10-year survival probability of only 3.4% (95% confidence interval CI = 0.5% to 23.3%, two-sided P = .002). Amplifications of regions not encompassing the MYCN locus were detected in 18.1% of patients and were associated with a 10-year survival probability of only 5.8% (95% CI = 1.5% to 22.2%, two-sided P < .001).
Using a unique large copy number data set of high-risk neuroblastoma cases, we identified a small subset of high-risk neuroblastoma patients with extremely low survival probability that might be eligible for inclusion in clinical trials of new therapeutics. The amplicons may also nominate alternative treatments that target the amplified genes.
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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