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1.
Commun Med (Lond) ; 4(1): 89, 2024 May 17.
Article in English | MEDLINE | ID: mdl-38760583

ABSTRACT

BACKGROUND: Despite the advent of neoadjuvant chemoradiotherapy (CRT), overall survival rates of esophageal adenocarcinoma (EAC) remain low. A readily induced mesenchymal transition of EAC cells contributes to resistance to CRT. METHODS: In this study, we aimed to chart the heterogeneity in cell state transition after CRT and to identify its underpinnings. A panel of 12 esophageal cultures were treated with CRT and ranked by their relative epithelial-mesenchymal plasticity. RNA-sequencing was performed on 100 pre-treatment biopsies. After RNA-sequencing, Ridge regression analysis was applied to correlate gene expression to ranked plasticity, and models were developed to predict mesenchymal transitions in patients. Plasticity score predictions of the three highest significant predictive models were projected on the pre-treatment biopsies and related to clinical outcome data. Motif enrichment analysis of the genes associated with all three models was performed. RESULTS: This study reveals NANOG as the key associated transcription factor predicting mesenchymal plasticity in EAC. Expression of NANOG in pre-treatment biopsies is highly associated with poor response to neoadjuvant chemoradiation, the occurrence of recurrences, and median overall survival difference in EAC patients (>48 months). Perturbation of NANOG reduces plasticity and resensitizes cell lines, organoid cultures, and patient-derived in vivo grafts. CONCLUSIONS: In conclusion, NANOG is a key transcription factor in mesenchymal plasticity in EAC and a promising predictive marker for outcome.


Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Although chemotherapy combined with radiotherapy (chemoradiotherapy) followed by surgery has improved survival, tumor recurrence and metastatic disease (that has spread to other parts of the body) are often observed after several months. In this study, we assessed the effect of chemoradiotherapy on esophageal cells in the lab to predict the effect in patients with esophageal cancer. To investigate this, genes were assessed from 12 different cell lines and 100 patient tissues. We revealed that levels of one of the genes, NANOG, associates with poor response in patients. NANOG could be a promising marker to predict outcome in patients with esophageal cancer. This knowledge might help clinicians to treat patients with esophageal cancer appropriately, or may lead to new or optimized treatments.

2.
Cell Rep Med ; 5(5): 101523, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38670098

ABSTRACT

Peritoneal metastases (PMs) from colorectal cancer (CRC) respond poorly to treatment and are associated with unfavorable prognosis. For example, the addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery in resectable patients shows limited benefit, and novel treatments are urgently needed. The majority of CRC-PMs represent the CMS4 molecular subtype of CRC, and here we queried the vulnerabilities of this subtype in pharmacogenomic databases to identify novel therapies. This reveals the copper ionophore elesclomol (ES) as highly effective against CRC-PMs. ES exhibits rapid cytotoxicity against CMS4 cells by targeting mitochondria. We find that a markedly reduced mitochondrial content in CMS4 cells explains their vulnerability to ES. ES demonstrates efficacy in preclinical models of PMs, including CRC-PMs and ovarian cancer organoids, mouse models, and a HIPEC rat model of PMs. The above proposes ES as a promising candidate for the local treatment of CRC-PMs, with broader implications for other PM-prone cancers.


Subject(s)
Colorectal Neoplasms , Mitochondria , Peritoneal Neoplasms , Colorectal Neoplasms/pathology , Colorectal Neoplasms/drug therapy , Peritoneal Neoplasms/secondary , Peritoneal Neoplasms/drug therapy , Peritoneal Neoplasms/therapy , Animals , Humans , Mitochondria/metabolism , Mitochondria/drug effects , Mice , Cell Line, Tumor , Rats , Female , Hyperthermic Intraperitoneal Chemotherapy/methods
3.
FEBS J ; 290(24): 5811-5834, 2023 12.
Article in English | MEDLINE | ID: mdl-37646174

ABSTRACT

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. While γ-secretase cleavage is necessary, we demonstrate it is insufficient for Notch activation and requires vesicular trafficking. Here, we report Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Dmt1-deficient cells are defective in Notch signalling and have perturbed endolysosomal trafficking and function. Dmt1 encodes for two isoforms, with and without an iron response element (ire). We show that isoform-specific silencing of Dmt1-ire and Dmt1+ire has opposite consequences on Notch-dependent cell fates in cell lines and intestinal organoids. Loss of Dmt1-ire suppresses Notch activation and promotes differentiation, whereas loss of Dmt1+ire causes Notch activation and maintains stem-progenitor cell fates. Dmt1 isoform expression correlates with Notch and Wnt signalling in Apc-deficient intestinal organoids and human colorectal cancers. Consistently, Dmt1-ire silencing induces Notch-dependent differentiation in colorectal cancer cells. These data identify Dmt1 isoforms as binary switches controlling Notch cell fate decisions in normal and tumour cells.


Subject(s)
Amyloid Precursor Protein Secretases , Cation Transport Proteins , Iron , Humans , Amyloid Precursor Protein Secretases/metabolism , Cell Line , Iron/metabolism , Iron-Binding Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Cation Transport Proteins/genetics , Regulatory Sequences, Nucleic Acid
4.
Cell Rep ; 41(10): 111761, 2022 12 06.
Article in English | MEDLINE | ID: mdl-36476851

ABSTRACT

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.


Subject(s)
Sarcoma, Ewing , Humans , Sarcoma, Ewing/genetics , Multiomics , Oncogenes , Cell Line , Transcription Factors
5.
Cell Rep Med ; 3(11): 100802, 2022 11 15.
Article in English | MEDLINE | ID: mdl-36334593

ABSTRACT

Neoadjuvant chemoradiotherapy (nCRT) improves outcomes in resectable esophageal adenocarcinoma (EAC), but acquired resistance precludes long-term efficacy. Here, we delineate these resistance mechanisms. RNA sequencing on matched patient samples obtained pre-and post-neoadjuvant treatment reveal that oxidative phosphorylation was the most upregulated of all biological programs following nCRT. Analysis of patient-derived models confirms that mitochondrial content and oxygen consumption strongly increase in response to nCRT and that ionizing radiation is the causative agent. Bioinformatics identifies estrogen-related receptor alpha (ESRRA) as the transcription factor responsible for reprogramming, and overexpression and silencing of ESRRA functionally confirm that its downstream metabolic rewiring contributes to resistance. Pharmacological inhibition of ESRRA successfully sensitizes EAC organoids and patient-derived xenografts to radiation. In conclusion, we report a profound metabolic rewiring following chemoradiation and demonstrate that its inhibition resensitizes EAC cells to radiation. These findings hold broader relevance for other cancer types treated with radiation as well.


Subject(s)
Drug Resistance, Neoplasm , Esophageal Neoplasms , Neoadjuvant Therapy , Organelle Biogenesis , Receptors, Estrogen , Humans , Esophageal Neoplasms/therapy , Mitochondria , Receptors, Estrogen/metabolism , Animals , ERRalpha Estrogen-Related Receptor
6.
BMC Biol ; 20(1): 182, 2022 08 19.
Article in English | MEDLINE | ID: mdl-35986286

ABSTRACT

BACKGROUND: SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn's disease (CD), suggesting a role in inflammation. RESULTS: We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte-to-inflammatory macrophage differentiation and lipopolysaccharide (LPS)-induced inflammatory activation, while inducing the generation of CD206+ regulatory macrophages that were shown to associate with a therapeutic response to anti-TNF in CD patients. SP140 preferentially occupies transcriptional start sites in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduces SP140 chromatin binding and thereby expression of SP140-regulated genes. GSK761 inhibits the expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal mucosa. CONCLUSIONS: This study identifies SP140 as a druggable epigenetic therapeutic target for CD.


Subject(s)
Crohn Disease , Tumor Necrosis Factor Inhibitors , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , Crohn Disease/genetics , Crohn Disease/metabolism , Cytokines/genetics , Cytokines/metabolism , Epigenesis, Genetic , Humans , Macrophages , Transcription Factors/genetics
8.
Cancer Res ; 82(3): 484-496, 2022 02 01.
Article in English | MEDLINE | ID: mdl-34853072

ABSTRACT

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.


Subject(s)
Anaplastic Lymphoma Kinase/antagonists & inhibitors , Neuroblastoma/genetics , Cell Line, Tumor , Humans , Neuroblastoma/pathology
9.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Article in English | MEDLINE | ID: mdl-34479993

ABSTRACT

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.


Subject(s)
DNA Repair/genetics , Guanosine/analogs & derivatives , Neuroblastoma/genetics , Adenine/metabolism , Child , Cytosine/metabolism , DNA Damage , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , Female , Guanine/metabolism , Guanosine/genetics , Guanosine/metabolism , Humans , Male , Mutagenesis , Neoplasm Recurrence, Local/genetics , Neuroblastoma/metabolism , Oxidative Stress , Polymorphism, Single Nucleotide/genetics
10.
Cancers (Basel) ; 13(1)2021 Jan 05.
Article in English | MEDLINE | ID: mdl-33466359

ABSTRACT

mRNA RT-qPCR is shown to be a very sensitive technique to detect minimal residual disease (MRD) in patients with neuroblastoma. Multiple mRNA markers are known to detect heterogeneous neuroblastoma cells in bone marrow (BM) or blood from patients. However, the limited volumes of BM and blood available can hamper the detection of multiple markers. To make optimal use of these samples, we developed a multiplex RT-qPCR for the detection of MRD in neuroblastoma. GUSB and PHOX2B were tested as single markers. The adrenergic markers TH, GAP43, CHRNA3 and DBH and mesenchymal markers POSTN, PRRX1 and FMO3 were tested in multiplex. Using control blood and BM, we established new thresholds for positivity. Comparison of multiplex and singleplex RT-qPCR results from 21 blood and 24 BM samples from neuroblastoma patients demonstrated a comparable sensitivity. With this multiplex RT-qPCR, we are able to test seven different neuroblastoma mRNA markers, which overcomes tumor heterogeneity and improves sensitivity of MRD detection, even in those samples of low RNA quantity. With resources and time being saved, reduction in sample volume and consumables can assist in the introduction of MRD by RT-qPCR into clinical practice.

11.
Nature ; 576(7786): 274-280, 2019 12.
Article in English | MEDLINE | ID: mdl-31802000

ABSTRACT

Embryonal tumours with multilayered rosettes (ETMRs) are aggressive paediatric embryonal brain tumours with a universally poor prognosis1. Here we collected 193 primary ETMRs and 23 matched relapse samples to investigate the genomic landscape of this distinct tumour type. We found that patients with tumours in which the proposed driver C19MC2-4 was not amplified frequently had germline mutations in DICER1 or other microRNA-related aberrations such as somatic amplification of miR-17-92 (also known as MIR17HG). Whole-genome sequencing revealed that tumours had an overall low recurrence of single-nucleotide variants (SNVs), but showed prevalent genomic instability caused by widespread occurrence of R-loop structures. We show that R-loop-associated chromosomal instability can be induced by the loss of DICER1 function. Comparison of primary tumours and matched relapse samples showed a strong conservation of structural variants, but low conservation of SNVs. Moreover, many newly acquired SNVs are associated with a mutational signature related to cisplatin treatment. Finally, we show that targeting R-loops with topoisomerase and PARP inhibitors might be an effective treatment strategy for this deadly disease.


Subject(s)
MicroRNAs/genetics , Neoplasms, Germ Cell and Embryonal/genetics , DEAD-box RNA Helicases/genetics , DNA Topoisomerases, Type I/genetics , Humans , Mutation , Neoplasms, Germ Cell and Embryonal/diagnosis , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , Polymorphism, Single Nucleotide , RNA, Long Noncoding , Recurrence , Ribonuclease III/genetics
12.
Nat Commun ; 10(1): 1530, 2019 04 04.
Article in English | MEDLINE | ID: mdl-30948783

ABSTRACT

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.


Subject(s)
Adrenergic Neurons/pathology , Cellular Reprogramming/genetics , Mesenchymal Stem Cells/pathology , Neuroblastoma/pathology , Receptor, Notch3/physiology , Adrenergic Neurons/metabolism , Cell Line, Tumor , Epigenesis, Genetic , Feedback, Physiological , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Mesenchymal Stem Cells/metabolism , Neuroblastoma/metabolism , Receptor, Notch3/genetics , Receptor, Notch3/metabolism
13.
Cancer Res ; 78(21): 6297-6307, 2018 11 01.
Article in English | MEDLINE | ID: mdl-30115695

ABSTRACT

Mutations affecting the RAS-MAPK pathway frequently occur in relapsed neuroblastoma tumors, which suggests that activation of this pathway is associated with a more aggressive phenotype. To explore this hypothesis, we generated several model systems to define a neuroblastoma RAS-MAPK pathway signature. Activation of this pathway in primary tumors indeed correlated with poor survival and was associated with known activating mutations in ALK and other RAS-MAPK pathway genes. Integrative analysis showed that mutations in PHOX2B, CIC, and DMD were also associated with an activated RAS-MAPK pathway. Mutation of PHOX2B and deletion of CIC in neuroblastoma cell lines induced activation of the RAS-MAPK pathway. This activation was independent of phosphorylated ERK in CIC knockout systems. Furthermore, deletion of CIC caused a significant increase in tumor growth in vivo These results show that the RAS-MAPK pathway is involved in tumor progression and establish CIC as a powerful tumor suppressor that functions downstream of this pathway in neuroblastoma.Significance: This work identifies CIC as a powerful tumor suppressor affecting the RAS-MAPK pathway in neuroblastoma and reinforces the importance of mutation-driven activation of this pathway in cancer. Cancer Res; 78(21); 6297-307. ©2018 AACR.


Subject(s)
MAP Kinase Signaling System , Neuroblastoma/genetics , Repressor Proteins/genetics , Animals , Cell Line, Tumor , Cluster Analysis , Disease Progression , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, ras , Genome, Human , Genomics , Homeodomain Proteins/metabolism , Humans , Mice , Mice, Knockout , Mice, Nude , Mutation , Neoplasm Recurrence, Local/genetics , Neoplasm Transplantation , Neuroblastoma/pathology , Phenotype , Phosphorylation , Prognosis , Repressor Proteins/metabolism , Signal Transduction , Transcription Factors/metabolism , Treatment Outcome
14.
Oncotarget ; 9(28): 19490-19507, 2018 Apr 13.
Article in English | MEDLINE | ID: mdl-29731961

ABSTRACT

Primary human colorectal tumors with a high stromal content have an increased capacity to metastasize. Cancer-associated fibroblasts (CAFs) promote metastasis, but the contribution of other stromal cell types is unclear. Here we searched for additional stromal cell types that contribute to aggressive tumor cell behavior. By making use of the 'immunome compendium'-a collection of gene signatures reflecting the presence of specific immune cell-types-we show that macrophage signatures are most strongly associated with a high CAF content and with poor prognosis in multiple large cohorts of primary tumors and liver metastases. Co-culturing macrophages with patient-derived colonospheres promoted 'budding' of small clusters of tumor cells from the bulk. Immunohistochemistry showed that budding tumor clusters in stroma-rich areas of T1 colorectal carcinomas were surrounded by macrophages. In vitro budding was accompanied by reduced levels of the tight junction protein occludin, but OCLN mRNA levels did not change, nor did markers of epithelial mesenchymal transition. Budding was accompanied by nuclear accumulation of ß-catenin, which was also observed in budding tumor cell clusters in situ. The NFκB inhibitor Sanguinarine resulted in a decrease in MMP7 protein expression and both NFκB inhibitor Sanguinarine and MMP inhibitor Batimastat prevented occludin degradation and budding. We conclude that macrophages contribute to the aggressive nature of stroma-rich colon tumors by promoting an MMP-dependent pathway that operates in parallel to classical EMT and leads to tight junction disruption.

15.
Nat Genet ; 49(8): 1261-1266, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28650485

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.


Subject(s)
Cell Differentiation/genetics , Epigenesis, Genetic , Neuroblastoma/genetics , Neuroblastoma/pathology , AC133 Antigen/genetics , Adrenergic Neurons/cytology , Cell Line, Tumor , Cell Lineage , Homeodomain Proteins/genetics , Humans , Mesoderm/cytology , Transcription Factors/metabolism , Transcriptome
16.
Nat Genet ; 47(12): 1411-4, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26523776

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Neoplastic , Gene Rearrangement , Neuroblastoma/genetics , Neuroblastoma/pathology , Telomerase/genetics , Telomere/genetics , DNA Helicases/genetics , Gene Amplification , Gene Deletion , Genome, Human , High-Throughput Nucleotide Sequencing/methods , Humans , N-Myc Proto-Oncogene Protein , Nuclear Proteins/genetics , Oncogene Proteins/genetics , X-linked Nuclear Protein
17.
Nat Genet ; 47(8): 864-71, 2015 08.
Article in English | MEDLINE | ID: mdl-26121087

ABSTRACT

The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.


Subject(s)
MAP Kinase Signaling System/genetics , Mitogen-Activated Protein Kinases/genetics , Mutation , Neoplasm Recurrence, Local/genetics , Neuroblastoma/genetics , ras Proteins/genetics , Anaplastic Lymphoma Kinase , Animals , Benzimidazoles/pharmacology , Blotting, Western , Cell Line, Tumor , Child , Child, Preschool , Chromosome Aberrations , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Infant , Male , Mice, SCID , Mitogen-Activated Protein Kinases/metabolism , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Phosphorylation/drug effects , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor Assays , ras Proteins/metabolism
18.
J Mol Diagn ; 17(1): 43-52, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25445214

ABSTRACT

PCR-based detection of minimal residual disease (MRD) in neuroblastoma is currently based on RNA markers; however, expression of these targets can vary, and only paired-like homeobox 2b has no background expression. We investigated whether chromosomal breakpoints, identified by whole-genome sequencing (WGS), can be used as patient-specific DNA MRD markers. WGS data were used to develop large numbers of real-time PCRs specific for tumors of eight patients. These PCRs were used to quantify chromosomal breakpoints in primary tumor and bone marrow samples. Finally, the DNA breakpoints with the highest abundance were compared with a panel of RNA markers. By WGS we identified 42 chromosomal breakpoints in tumor samples from eight patients and developed specific quantitative real-time PCRs for each breakpoint. The tumor-specific breakpoints were all present in bone marrow at diagnosis. For one patient slight clonal selection was observed in response to treatment. Positivity of DNA MRD markers preceded disease progression in four of five patients; in one patient the RNA markers remained negative. For 16 of 22 samples MRD levels determined by RNA and DNA were comparable and in 6 of 22 samples higher MRD levels were detected by DNA markers. DNA breakpoints used as MRD targets in neuroblastoma are reliable and stable markers. In addition, this technique might be applicable for detecting tumor cells in other types of cancer.


Subject(s)
Biomarkers, Tumor/genetics , Chromosome Breakpoints , Neoplasm, Residual/genetics , Neuroblastoma/genetics , Real-Time Polymerase Chain Reaction/methods , Antineoplastic Agents/therapeutic use , Bone Marrow/metabolism , Bone Marrow/pathology , Child , Child, Preschool , Female , Genetic Markers , Genome, Human , Genome-Wide Association Study , High-Throughput Nucleotide Sequencing , Humans , Infant , Male , Neoplasm Staging , Neoplasm, Residual/drug therapy , Neoplasm, Residual/mortality , Neoplasm, Residual/pathology , Neuroblastoma/drug therapy , Neuroblastoma/mortality , Neuroblastoma/pathology , Survival Analysis
19.
Eur J Cancer ; 50(3): 628-37, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24321263

ABSTRACT

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models.


Subject(s)
Cell Line, Tumor , Neuroblastoma/genetics , Neuroblastoma/pathology , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Child , Child, Preschool , Culture Media, Serum-Free , Genotype , Humans , Infant , Mice , Mice, Nude , Neuroblastoma/metabolism , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism
20.
Acta Neuropathol ; 125(3): 385-94, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23179372

ABSTRACT

Recent studies showed frequent mutations in histone H3 lysine 27 (H3K27) demethylases in medulloblastomas of Group 3 and Group 4, suggesting a role for H3K27 methylation in these tumors. Indeed, trimethylated H3K27 (H3K27me3) levels were shown to be higher in Group 3 and 4 tumors compared to WNT and SHH medulloblastomas, also in tumors without detectable mutations in demethylases. Here, we report that polycomb genes, required for H3K27 methylation, are consistently upregulated in Group 3 and 4 tumors. These tumors show high expression of the homeobox transcription factor OTX2. Silencing of OTX2 in D425 medulloblastoma cells resulted in downregulation of polycomb genes such as EZH2, EED, SUZ12 and RBBP4 and upregulation of H3K27 demethylases KDM6A, KDM6B, JARID2 and KDM7A. This was accompanied by decreased H3K27me3 and increased H3K27me1 levels in promoter regions. Strikingly, the decrease of H3K27me3 was most prominent in promoters that bind OTX2. OTX2-bound promoters showed high levels of the H3K4me3 and H3K9ac activation marks and intermediate levels of the H3K27me3 inactivation mark, reminiscent of a bivalent modification. After silencing of OTX2, H3K27me3 levels strongly dropped, but H3K4me3 and H3K9ac levels remained high. OTX2-bound bivalent genes showed high expression levels in D425, but the expression of most of these genes did not change after OTX2 silencing and loss of the H3K27me3 mark. Maintaining promoters in a bivalent state by sustaining H3K27 trimethylation therefore seems to be an important function of OTX2 in medulloblastoma, while other transcription factors might regulate the actual expression levels of these genes.


Subject(s)
Gene Expression Regulation, Neoplastic/physiology , Histones/metabolism , Otx Transcription Factors/genetics , Otx Transcription Factors/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Gene Expression Profiling , Histones/genetics , Humans , Medulloblastoma/classification , Medulloblastoma/genetics , Medulloblastoma/pathology , Methylation , Oligonucleotide Array Sequence Analysis , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolism , Promoter Regions, Genetic/genetics , RNA, Messenger
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