Publications by authors named "Katleen De Preter"

110 Publications

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Genes Chromosomes Cancer 2021 Apr 9;60(4):272-281. Epub 2021 Jan 9.

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.
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http://dx.doi.org/10.1002/gcc.22931DOI Listing
April 2021

Author Correction: Benchmarking of cell type deconvolution pipelines for transcriptomics data.

Nat Commun 2020 Dec 2;11(1):6291. Epub 2020 Dec 2.

Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20288-9.
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http://dx.doi.org/10.1038/s41467-020-20288-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710698PMC
December 2020

Benchmarking of cell type deconvolution pipelines for transcriptomics data.

Nat Commun 2020 11 6;11(1):5650. Epub 2020 Nov 6.

Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Many computational methods have been developed to infer cell type proportions from bulk transcriptomics data. However, an evaluation of the impact of data transformation, pre-processing, marker selection, cell type composition and choice of methodology on the deconvolution results is still lacking. Using five single-cell RNA-sequencing (scRNA-seq) datasets, we generate pseudo-bulk mixtures to evaluate the combined impact of these factors. Both bulk deconvolution methodologies and those that use scRNA-seq data as reference perform best when applied to data in linear scale and the choice of normalization has a dramatic impact on some, but not all methods. Overall, methods that use scRNA-seq data have comparable performance to the best performing bulk methods whereas semi-supervised approaches show higher error values. Moreover, failure to include cell types in the reference that are present in a mixture leads to substantially worse results, regardless of the previous choices. Altogether, we evaluate the combined impact of factors affecting the deconvolution task across different datasets and propose general guidelines to maximize its performance.
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http://dx.doi.org/10.1038/s41467-020-19015-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648640PMC
November 2020

Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome.

Epigenetics 2020 Oct 19:1-11. Epub 2020 Oct 19.

Cancer Research Institute Ghent (CRIG) , Ghent, Belgium.

The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulphite-based cfDNA methylation profiling. Fifteen tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulphite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics. All preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes. The methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.
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http://dx.doi.org/10.1080/15592294.2020.1827714DOI Listing
October 2020

When DNA gets in the way: A cautionary note for DNA contamination in extracellular RNA-seq studies.

Proc Natl Acad Sci U S A 2020 08;117(32):18934-18936

Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium.

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http://dx.doi.org/10.1073/pnas.2001675117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431080PMC
August 2020

Minimally invasive classification of paediatric solid tumours using reduced representation bisulphite sequencing of cell-free DNA: a proof-of-principle study.

Epigenetics 2021 Jan-Feb;16(2):196-208. Epub 2020 Jul 14.

Department of Biomolecular Medicine, Ghent University , Ghent, Belgium.

In the clinical management of paediatric solid tumours, histological examination of tumour tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumour is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumour samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of paediatric tumours using cell-free reduced representation bisulphite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumour type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in paediatric oncology in a cost-effective and minimally invasive manner.
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http://dx.doi.org/10.1080/15592294.2020.1790950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889189PMC
July 2020

Correction to: The pitfalls and promise of liquid biopsies for diagnosing and treating solid tumors in children: as review.

Eur J Pediatr 2020 09;179(9):1497-1498

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

In the original version of this article, a reader pointed out that there was a mistake in the phrasing in a paragraph. This could potentially be harmful to children. The authors agree to change the wording. "vitreous fluid" will be changed to "aqueous humor".
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http://dx.doi.org/10.1007/s00431-020-03692-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645481PMC
September 2020

Distinct Notch1 and BCL11B requirements mediate human γδ/αβ T cell development.

EMBO Rep 2020 05 7;21(5):e49006. Epub 2020 Apr 7.

Department of Diagnostic Sciences, Ghent University, Ghent, Belgium.

γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T-lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αβ-lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR-17-92 cluster is a Notch1 target in immature thymocytes and that miR-17 can restrict BCL11B expression in these Notch-dependent T cell precursors. We show that enforced miR-17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αβ but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage-specific Notch-driven negative feedback loop through which miR-17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease-associated genes BCL11B and the miR-17-92 cluster in a human context.
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http://dx.doi.org/10.15252/embr.201949006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202205PMC
May 2020

The ETS transcription factor ETV5 is a target of activated ALK in neuroblastoma contributing to increased tumour aggressiveness.

Sci Rep 2020 01 14;10(1):218. Epub 2020 Jan 14.

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Neuroblastoma is an aggressive childhood cancer arising from sympatho-adrenergic neuronal progenitors. The low survival rates for high-risk disease point to an urgent need for novel targeted therapeutic approaches. Detailed molecular characterization of the neuroblastoma genomic landscape indicates that ALK-activating mutations are present in 10% of primary tumours. Together with other mutations causing RAS/MAPK pathway activation, ALK mutations are also enriched in relapsed cases and ALK activation was shown to accelerate MYCN-driven tumour formation through hitherto unknown ALK-driven target genes. To gain further insight into how ALK contributes to neuroblastoma aggressiveness, we searched for known oncogenes in our previously reported ALK-driven gene signature. We identified ETV5, a bona fide oncogene in prostate cancer, as robustly upregulated in neuroblastoma cells harbouring ALK mutations, and show high ETV5 levels downstream of the RAS/MAPK axis. Increased ETV5 expression significantly impacted migration, invasion and colony formation in vitro, and ETV5 knockdown reduced proliferation in a murine xenograft model. We also established a gene signature associated with ETV5 knockdown that correlates with poor patient survival. Taken together, our data highlight ETV5 as an intrinsic component of oncogenic ALK-driven signalling through the MAPK axis and propose that ETV5 upregulation in neuroblastoma may contribute to tumour aggressiveness.
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http://dx.doi.org/10.1038/s41598-019-57076-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959226PMC
January 2020

The pitfalls and promise of liquid biopsies for diagnosing and treating solid tumors in children: a review.

Eur J Pediatr 2020 Feb 3;179(2):191-202. Epub 2020 Jan 3.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Cell-free DNA profiling using patient blood is emerging as a non-invasive complementary technique for cancer genomic characterization. Since these liquid biopsies will soon be integrated into clinical trial protocols for pediatric cancer treatment, clinicians should be informed about potential applications and advantages but also weaknesses and potential pitfalls. Small retrospective studies comparing genetic alterations detected in liquid biopsies with tumor biopsies for pediatric solid tumor types are encouraging. Molecular detection of tumor markers in cell-free DNA could be used for earlier therapy response monitoring and residual disease detection as well as enabling detection of pathognomonic and therapeutically relevant genomic alterations.Conclusion: Existing analyses of liquid biopsies from children with solid tumors increasingly suggest a potential relevance for molecular diagnostics, prognostic assessment, and therapeutic decision-making. Gaps remain in the types of tumors studied and value of detection methods applied. Here we review the current stand of liquid biopsy studies for pediatric solid tumors with a dedicated focus on cell-free DNA analysis. There is legitimate hope that integrating fully validated liquid biopsy-based innovations into the standard of care will advance patient monitoring and personalized treatment of children battling solid cancers.What is Known:• Liquid biopsies are finding their way into routine oncological screening, diagnosis, and disease monitoring in adult cancer types fast.• The most widely adopted source for liquid biopsies is blood although other easily accessible body fluids, such as saliva, pleural effusions, urine, or cerebrospinal fluid (CSF) can also serve as sources for liquid biopsiesWhat is New:• Retrospective proof-of-concept studies in small cohorts illustrate that liquid biopsies in pediatric solid tumors yield tremendous potential to be used in diagnostics, for therapy response monitoring and in residual disease detection.• Liquid biopsy diagnostics could tackle some long-standing issues in the pediatric oncology field; they can enable accurate genetic diagnostics in previously unbiopsied tumor types like renal tumors or brain stem tumors leading to better treatment strategies.
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http://dx.doi.org/10.1007/s00431-019-03545-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971142PMC
February 2020

Author Correction: Integrative analysis identifies lincRNAs up- and downstream of neuroblastoma driver genes.

Sci Rep 2019 Jul 17;9(1):10536. Epub 2019 Jul 17.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-46785-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635357PMC
July 2019

Publisher Correction: In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors.

Sci Rep 2019 Jun 4;9(1):8360. Epub 2019 Jun 4.

Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-44435-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547841PMC
June 2019

DREAM target reactivation by core transcriptional regulators supports neuroblastoma growth.

Mol Cell Oncol 2019 3;6(2):1565470. Epub 2019 Feb 3.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Chromosome 17q gains are a common alteration in high-risk neuroblastomas with unknown functional significance. We identified a 17q super-enhancer regulated T-box Transcription Factor 2 (TBX2) as constituent of a core regulatory circuitry driving proliferation through enhancing V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived (avian) (MYCN)/Forkhead box protein M1(FOXM1) reactivation of dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) targets, which can be affected synergistically by combined cyclin-dependent kinase 7 and Bromo-domain inhibition.
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http://dx.doi.org/10.1080/23723556.2019.1565470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512935PMC
February 2019

Integrative analysis identifies lincRNAs up- and downstream of neuroblastoma driver genes.

Sci Rep 2019 04 5;9(1):5685. Epub 2019 Apr 5.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

Long intergenic non-coding RNAs (lincRNAs) are emerging as integral components of signaling pathways in various cancer types. In neuroblastoma, only a handful of lincRNAs are known as upstream regulators or downstream effectors of oncogenes. Here, we exploit RNA sequencing data of primary neuroblastoma tumors, neuroblast precursor cells, neuroblastoma cell lines and various cellular perturbation model systems to define the neuroblastoma lincRNome and map lincRNAs up- and downstream of neuroblastoma driver genes MYCN, ALK and PHOX2B. Each of these driver genes controls the expression of a particular subset of lincRNAs, several of which are associated with poor survival and are differentially expressed in neuroblastoma tumors compared to neuroblasts. By integrating RNA sequencing data from both primary tumor tissue and cancer cell lines, we demonstrate that several of these lincRNAs are expressed in stromal cells. Deconvolution of primary tumor gene expression data revealed a strong association between stromal cell composition and driver gene status, resulting in differential expression of these lincRNAs. We also explored lincRNAs that putatively act upstream of neuroblastoma driver genes, either as presumed modulators of driver gene activity, or as modulators of effectors regulating driver gene expression. This analysis revealed strong associations between the neuroblastoma lincRNAs MIAT and MEG3 and MYCN and PHOX2B activity or expression. Together, our results provide a comprehensive catalogue of the neuroblastoma lincRNome, highlighting lincRNAs up- and downstream of key neuroblastoma driver genes. This catalogue forms a solid basis for further functional validation of candidate neuroblastoma lincRNAs.
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http://dx.doi.org/10.1038/s41598-019-42107-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451017PMC
April 2019

ALK positively regulates MYCN activity through repression of HBP1 expression.

Oncogene 2019 04 11;38(15):2690-2705. Epub 2018 Dec 11.

Center for Medical Genetics Ghent (CMGG), Ghent University, Ghent, Belgium.

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PIK-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PIK antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.
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http://dx.doi.org/10.1038/s41388-018-0595-3DOI Listing
April 2019

Circulating microRNA biomarkers for metastatic disease in neuroblastoma patients.

JCI Insight 2018 12 6;3(23). Epub 2018 Dec 6.

Center for Medical Genetics, Department of Biomolecular Medicine, and.

In this study, the circulating miRNome from diagnostic neuroblastoma serum was assessed for identification of noninvasive biomarkers with potential in monitoring metastatic disease. After determining the circulating neuroblastoma miRNome, 743 miRNAs were screened in 2 independent cohorts of 131 and 54 patients. Evaluation of serum miRNA variance in a model testing for tumor stage, MYCN status, age at diagnosis, and overall survival revealed tumor stage as the most significant factor impacting miRNA abundance in neuroblastoma serum. Differential abundance analysis between patients with metastatic and localized disease revealed 9 miRNAs strongly associated with metastatic stage 4 disease in both patient cohorts. Increasing levels of these miRNAs were also observed in serum from xenografted mice bearing human neuroblastoma tumors. Moreover, murine serum miRNA levels were strongly associated with tumor volume. These findings were validated in longitudinal serum samples from metastatic neuroblastoma patients, where the 9 miRNAs were associated with disease burden and treatment response.
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http://dx.doi.org/10.1172/jci.insight.97021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328024PMC
December 2018

In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors.

Sci Rep 2018 11 30;8(1):17468. Epub 2018 Nov 30.

Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium.

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway.
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http://dx.doi.org/10.1038/s41598-018-35868-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269481PMC
November 2018

Integrated proximal proteomics reveals IRS2 as a determinant of cell survival in ALK-driven neuroblastoma.

Sci Signal 2018 11 20;11(557). Epub 2018 Nov 20.

Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.

Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry-based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI-sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis.
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http://dx.doi.org/10.1126/scisignal.aap9752DOI Listing
November 2018

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets.

Nat Commun 2018 11 19;9(1):4866. Epub 2018 Nov 19.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.
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http://dx.doi.org/10.1038/s41467-018-06699-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242972PMC
November 2018

Meta-mining of copy number profiles of high-risk neuroblastoma tumors.

Sci Data 2018 10 30;5:180240. Epub 2018 Oct 30.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Neuroblastoma, a pediatric tumor of the sympathetic nervous system, is predominantly driven by copy number aberrations, which predict survival outcome in global neuroblastoma cohorts and in low-risk cases. For high-risk patients there is still a need for better prognostic biomarkers. Via an international collaboration, we collected copy number profiles of 556 high-risk neuroblastomas generated on different array platforms. This manuscript describes the composition of the dataset, the methods used to process the data, including segmentation and aberration calling, and data validation. t-SNE analysis shows that samples cluster according to MYCN status, and shows a difference between array platforms. 97.3% of samples are characterized by the presence of segmental aberrations, in regions frequently affected in neuroblastoma. Focal aberrations affect genes known to be involved in neuroblastoma, such as ALK and LIN28B. To conclude, we compiled a unique large copy number dataset of high-risk neuroblastoma tumors, available via R2 and a Shiny web application. The availability of patient survival data allows to further investigate the prognostic value of copy number aberrations.
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http://dx.doi.org/10.1038/sdata.2018.240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207068PMC
October 2018

Correction: Mapping the genomic landscape of inherited retinal disease genes prioritizes genes prone to coding and noncoding copy-number variations.

Genet Med 2019 Aug;21(8):1998

Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.

The original version of this Article contained an error in the spelling of the author Anja K. Mayer, which was incorrectly given as Anja Kathrin Mayer. This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41436-018-0305-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609298PMC
August 2019

LDHA in Neuroblastoma Is Associated with Poor Outcome and Its Depletion Decreases Neuroblastoma Growth Independent of Aerobic Glycolysis.

Clin Cancer Res 2018 11 20;24(22):5772-5783. Epub 2018 Jun 20.

Department of Pediatrics and Adolescent Medicine, University Medical Center Ulm, Ulm, Germany.

To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB). Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated with survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity, and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model. Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity, and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed. High expression of LDHA is independently associated with outcome of NB, and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2578DOI Listing
November 2018

IncGraph: Incremental graphlet counting for topology optimisation.

PLoS One 2018 26;13(4):e0195997. Epub 2018 Apr 26.

Data Mining and Modelling for Biomedicine group, VIB Center for Inflammation Research, Ghent, Belgium.

Motivation: Graphlets are small network patterns that can be counted in order to characterise the structure of a network (topology). As part of a topology optimisation process, one could use graphlet counts to iteratively modify a network and keep track of the graphlet counts, in order to achieve certain topological properties. Up until now, however, graphlets were not suited as a metric for performing topology optimisation; when millions of minor changes are made to the network structure it becomes computationally intractable to recalculate all the graphlet counts for each of the edge modifications.

Results: IncGraph is a method for calculating the differences in graphlet counts with respect to the network in its previous state, which is much more efficient than calculating the graphlet occurrences from scratch at every edge modification made. In comparison to static counting approaches, our findings show IncGraph reduces the execution time by several orders of magnitude. The usefulness of this approach was demonstrated by developing a graphlet-based metric to optimise gene regulatory networks. IncGraph is able to quickly quantify the topological impact of small changes to a network, which opens novel research opportunities to study changes in topologies in evolving or online networks, or develop graphlet-based criteria for topology optimisation.

Availability: IncGraph is freely available as an open-source R package on CRAN (incgraph). The development version is also available on GitHub (rcannood/incgraph).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195997PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919487PMC
July 2018

Network Modeling of microRNA-mRNA Interactions in Neuroblastoma Tumorigenesis Identifies miR-204 as a Direct Inhibitor of MYCN.

Cancer Res 2018 06 2;78(12):3122-3134. Epub 2018 Apr 2.

Children's Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, Randwick, New South Wales, Australia.

Neuroblastoma is a pediatric cancer of the sympathetic nervous system where amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN mice to age-matched wild-type controls, we devised a predicted miRNA-mRNA interaction network. Among the miRNA-mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with -amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation and tumorigenesis Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis. Network modeling of miRNA-mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3034DOI Listing
June 2018

Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative role of HDX as a negative regulator.

Biochem Biophys Res Commun 2018 05 26;499(2):291-298. Epub 2018 Mar 26.

Department of Pediatrics, Clinical Sciences, Lund University, Lund, Sweden. Electronic address:

Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism.
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http://dx.doi.org/10.1016/j.bbrc.2018.03.150DOI Listing
May 2018

Genomic Amplifications and Distal 6q Loss: Novel Markers for Poor Survival in High-risk Neuroblastoma Patients.

J Natl Cancer Inst 2018 10;110(10):1084-1093

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Background: 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.

Methods: 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.

Results: 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).

Conclusions: 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.
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http://dx.doi.org/10.1093/jnci/djy022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186524PMC
October 2018

Cross-Cohort Analysis Identifies a TEAD4-MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma.

Cancer Discov 2018 05 6;8(5):582-599. Epub 2018 Mar 6.

Department of Systems Biology, Columbia University, New York, New York.

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with amplification. Silencing of either gene collapsed -amplified () neuroblastoma transcriptional hallmarks and abrogated viability and Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. .
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http://dx.doi.org/10.1158/2159-8290.CD-16-0861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967627PMC
May 2018

The mutational landscape of , and driven murine neuroblastoma mimics human disease.

Oncotarget 2018 Feb 22;9(9):8334-8349. Epub 2017 Dec 22.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems ( Th- Dbh- and ). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.
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http://dx.doi.org/10.18632/oncotarget.23614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823580PMC
February 2018

Computational deconvolution of transcriptomics data from mixed cell populations.

Bioinformatics 2018 06;34(11):1969-1979

Center for Medical Genetics Ghent (CMGG), Ghent University, 9000 Ghent, Belgium.

Summary: Gene expression analyses of bulk tissues often ignore cell type composition as an important confounding factor, resulting in a loss of signal from lowly abundant cell types. In this review, we highlight the importance and value of computational deconvolution methods to infer the abundance of different cell types and/or cell type-specific expression profiles in heterogeneous samples without performing physical cell sorting. We also explain the various deconvolution scenarios, the mathematical approaches used to solve them and the effect of data processing and different confounding factors on the accuracy of the deconvolution results.

Contact: katleen.depreter@ugent.be.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty019DOI Listing
June 2018

Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome.

Oncotarget 2017 Dec 6;8(63):106820-106832. Epub 2017 Nov 6.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Background: Neuroblastoma is an aggressive childhood malignancy of the sympathetic nervous system. Despite multi-modal therapy, survival of high-risk patients remains disappointingly low, underscoring the need for novel treatment strategies. The discovery of activating mutations opened the way to precision treatment in a subset of these patients. Previously, we investigated the transcriptional effects of pharmacological ALK inhibition on neuroblastoma cell lines, six hours after TAE684 administration, resulting in the 77-gene ALK signature, which was shown to gradually decrease from 120 minutes after TAE684 treatment, to gain deeper insight into the molecular effects of oncogenic ALK signaling.

Aim: Here, we further dissected the transcriptional dynamic profiles of neuroblastoma cells upon TAE684 treatment in a detailed timeframe of ten minutes up to six hours after inhibition, in order to identify additional early targets for combination treatment.

Results: We observed an unexpected initial upregulation of positively regulated MYCN target genes following subsequent downregulation of overall MYCN activity. In addition, we identified adrenomedullin (ADM), previously shown to be implicated in sunitinib resistance, as the earliest response gene upon ALK inhibition.

Conclusions: We describe the early and late effects of ALK inhibitor TAE684 treatment on the neuroblastoma transcriptome. The observed unexpected upregulation of ADM warrants further investigation in relation to putative ALK resistance in neuroblastoma patients currently undergoing ALK inhibitor treatment.
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http://dx.doi.org/10.18632/oncotarget.22423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739776PMC
December 2017