Biology subjects

Speleman, F.

Publications and source records attributed to Speleman, F..

17 recordsLinked to original sources

bioRxiv2026

SOX11 stimulates γδ T-cell differentiation and synergizes with LMO2 and MYCN to drive γδ-like T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous hematologic malignancy in which LMO2 {gamma}{delta}-like T-ALL represents a rare but clinically aggressive subtype associated with poor treatment response and inferior survival. Integrated transcriptomic analyses identified high SOX11 expression as a defining feature of high-risk LMO2 {gamma}{delta}-like T-ALL, where elevated SOX11 levels correlated with refractory disease and poor clinical outcome. To investigate the functional role of SOX11 in {gamma}{delta} T-cell biology and leukemogenesis, we generated a conditional R26-SOX11 mouse model enabling lineage-specific SOX11 overexpression in T-cell progenitors. SOX11 expression promoted expansion of the innate {gamma}{delta} T-cell compartment in thymus, spleen, and bone marrow, accompanied by transcriptional activation of {gamma}{delta} T-cell differentiation, activation, and cytotoxicity programs. However, SOX11 overexpression alone was insufficient to induce leukemia or confer thymocyte self-renewal capacity. In contrast, combined SOX11 and LMO2 overexpression markedly accelerated T-ALL development and strongly increased the incidence of {gamma}{delta}-like leukemias, thereby recapitulating the human high-risk LMO2 {gamma}{delta}-like T-ALL subtype. Mechanistically, SOX11 expanded the pre-leukemic DN3 thymocyte compartment in LMO2-driven mouse model while promoting differentiation toward the {gamma}{delta} lineage. Transcriptomic profiling identified activation of MYCN-associated transcriptional programs in SOX11/LMO2 pre-leukemic thymocytes. Consistently, MYCN was highly expressed in human LMO2 {gamma}{delta}-like T-ALL, and recurrent stabilizing MYCN P44L mutations were enriched in this subtype. Functional validation using genetic and transplantation-based mouse models demonstrated that SOX11 cooperates with MYCN to accelerate T-ALL onset. Together, these findings establish a cooperative SOX11-MYCN oncogenic axis driving {gamma}{delta}-like T-ALL and provide a novel preclinical model for investigating therapeutic vulnerabilities in this high-risk leukemia subtype.

cancer biology↗

bioRxiv2025

Widespread DNA off-targeting confounds studies of RNA chromatin occupancy

The importance of long noncoding RNA (lncRNA) functions is recognized across biological systems, but their modes of action remain poorly understood. One mechanism proposed to be particularly common is gene expression regulation via recruitment to specific genomic regions. Several high-throughput sequencing methods were developed for studying the genome-wide chromatin occupancy of lncRNAs, including ChIRP-seq, CHART-seq, and RAP-seq. These methods utilize biotin-labeled probes targeting the RNA of interest to isolate and recover the chromatin associated with it. Many of the datasets obtained with these methods contain thousands of binding sites, which appears to be in contradiction with the low abundance of the interrogated lncRNAs. We studied the chromatin interactome of NESPR lncRNA in cells with varying levels of endogenous expression and then performed a meta-analysis using dozens of RNA-chromatin interaction datasets in human and mouse cells. We demonstrate that thousands of regions reported to bind lncRNAs most likely arise from the spurious recovery of DNA elements, where the ends of the recovered DNA fragments exhibit partial complementarity with the probes used for the pulldown. In addition, crucial controls were rarely used in studies profiling RNA-chromatin interactions. Therefore, most chromatin regions reported as bound by trans-acting RNAs in recent studies in mammalian cells appear to be technical artifacts. We provide suggestions for assessing the quality of RNA-chromatin datasets and their improvement.

molecular biology↗

bioRxiv2025

AURKA promotes malignant properties of neuroblastoma and is downregulated by the PP2A pathway

The role of Aurora kinase A (AURKA) in malignant transformation of sympathoadrenergic progenitors (SAPs), the cells of origin of neuroblastoma (NB), and in progression and maintenance of NB is incompletely understood. MYCN is amplified in a subset of high-risk NB cases with poor prognosis. CDKN2A, which encodes the tumor suppressors p16INK4A and p14ARF, is deleted or silenced in a subset of NB. PPP2R4 (also known as PTPA) is a key activator of the phosphatase PP2A known to suppress NB. We show that AURKA and MYCN expression decreases postnatally in murine adrenal glands, suggesting that their expression may contribute to the maintenance of SAPs. In mouse embryonic fibroblasts (MEFs), used in place of difficult-to-isolate SAPs, AURKA contributed to transformation only when MYCN was overexpressed and INK4A/ARF was depleted. In transgenic mice expressing human AURKA (hAURKA) in adrenal glands, hAURKA mRNA was present but protein was undetectable, likely due to AURKAs short half-life, possibly limiting its role in initial transformation of SAPs. However, AURKA cooperated with MYCN to promote progression of established human SH-EP NB cells lacking INK4A/ARF. In addition, AURKA expression increased during NB progression in TH-MYCN mice and correlated with poor prognosis in human NB, supporting a role for AURKA in disease progression and maintenance. CDKN2A and PPP2R4 mRNA levels also rose during NB progression in TH-MYCN mice, possibly reflecting inadequate tumor-suppressive responses. Bayesian analysis of TH-MYCN expression data supported a role for PPP2R4 in inhibiting NB maintenance. Knockout of PPP2R4 in KELLY NB cells, along with reduced mePPP2CA, the catalytic subunit of PP2A, increased AURKA protein levels, indicating that PP2A regulates AURKA abundance. Together, these findings indicate that AURKA promotes malignant properties of NB and is decreased by the PP2A pathway in NB cells

cancer biology↗

bioRxiv2025

Stress attenuation by the adrenergic-specific lncRNA NESPR prevents cell death in neuroblastoma cells

Neuroblastoma is a pediatric cancer of the sympathetic nervous system characterized by heterogeneous cell states that mirror normal differentiation trajectories. Each state is governed by a core regulatory transcriptional circuitry that reinforces cell identity through an autoregulatory feedforward loop. We identified the long non-coding RNA NESPR as specifically expressed in adrenergic neuroblastoma cells. NESPR expression correlates with high-risk neuroblastoma clinical parameters and poor patient survival. NESPR is located within an insulated gene neighborhood alongside PHOX2B, a master transcription factor of the adrenergic identity. NESPR depletion reduced cell proliferation and increased caspase activity in neuroblastoma cell lines, and NESPR knockout in a neuroblastoma zebrafish model led to reduced tumor penetrance. Subcellular localization revealed NESPR to be a cytosolic long non-coding RNA, suggesting a trans-regulatory function. RNA-sequencing following NESPR depletion revealed a shift from an adrenergic to a mesenchymal cell state, due to proteotoxic stress-induced molecular reprogramming. These findings suggest NESPR as a regulator of neuroblastoma cell identity with potential therapeutic opportunities in high-risk neuroblastoma cases.

genetics↗

bioRxiv2025

Targeting replication stress in neuroblastoma by exploiting the synergistic potential of second generation RRM2 and CHK1 inhibitors

Tumor cells often cope with elevated levels of replication stress (RS) causing increased dependency on ATR-CHK1 signalling. We previously presented RRM2, the regulatory component of the ribonucleotide reductase (RNR) enzyme, as novel dependency in neuroblastoma, in keeping with its role in RS resistance. We identified strong synergism for combined RRM2-CHK1 inhibition using the iron chelator triapine and prexasertib respectively. To obtain direct RNR targeting, we evaluated a novel inhibitor, TAS1553, specifically disrupting the RNR complex in this study. Treatment with TAS1553 impedes cell growth and induces enhanced RS, DNA damage and apoptosis. We demonstrated strong synergism between TAS1553 the CHK1 inhibitors prexasertib and SRA737 in both NB and sarcoma cell lines, underscoring the broad clinical potential of combinatorial RRM2-CHK1 inhibition. Transcriptome profiling demonstrated strong overlap between the different RRM2-CHK1 treatments and revealed differential expression of RNA splicing components, opening new perspectives for combination treatments using splicing inhibitors. Altogether, this study paves the way for further preclinical testing of second generation RRM2 and CHK1 inhibitors such as TAS1553 and SRA737 in neuroblastoma and sarcomas.

cancer biology↗

bioRxiv2024

RUVBL1 and RUVBL2 are druggable MYCN regulators in neuroblastoma.

High-risk neuroblastoma is characterized by MYCN amplification and high MYCN or MYC gene expression. These patients have a poor prognosis and there is an urgent need for more effective drugs. While strategies to develop inhibitors that directly target the MYC proteins have remained largely unsuccessful, recent preclinical studies have identified ATR, a key protein of the DNA damage response, as a promising alternative therapeutic target. Here we identified a strong RUVBL1 and RUVBL2 signature in transcriptomics data derived from different MYCN-driven mice tumors treated with ATR inhibitors. The RUVBL proteins form a complex with ATPase activity that has broad cellular functions and we demonstrate that pharmacological inhibition of this protein complex results in a strong reduction of MYC signaling, cell cycle arrest, DNA damage and apoptosis. We confirmed the association with MYCN and identified the RUVBL genes as independent prognosticators in human primary neuroblastoma data.

cancer biology↗

bioRxiv2023

Preclinical exploration of the DNA Damage Response pathway using the interactive neuroblastoma cell line explorer CLEAN.

Neuroblastoma (NB) is the most common cancer in infancy with an urgent need for more efficient targeted therapies. The development of novel (combinatorial) treatment strategies relies on extensive explorations of signaling perturbations in neuroblastoma cell lines, using RNA-Seq or other high throughput technologies (e.g., phosphoproteomics). This typically requires dedicated bioinformatics support, which is not always available. Additionally, while data from published studies are highly valuable and raw data (e.g., fastq files) are nowadays released in public repositories, data processing is time-consuming and again difficult without bioinformatics support. To facilitate NB research, more user-friendly and immediately accessible platforms are needed to explore newly generated as well as existing high throughput data. To make this possible, we developed an interactive data centralization and visualization web application, called CLEAN (the Cell Line Explorer web Application of Neuroblastoma data; https://ccgg.ugent.be/shiny/clean/). By focusing on the regulation of the DNA damage response, a therapeutic target of major interest in neuroblastoma, we demonstrate how CLEAN can be used to gain novel mechanistic insights and identify putative drug targets in neuroblastoma.

cancer biology↗

bioRxiv2023

MOXD1 is a gate-keeper of organ homeostasis and functions as a tumor-suppressor in neuroblastoma

Neuroblastoma is a childhood cancer believed to result from dysfunctional development. Its origin during embryogenesis remains poorly understood. The lack of appropriate models has hindered in-depth mapping of tumor-driving events. Here, we identify a novel tumor-suppressor gene that predicts poor survival in high-risk disease, by applying bulk and single cell RNA sequencing data of neuroblastoma and human fetal adrenal glands. Trunk neural crest-specific MOXD1 discriminates cell populations during normal and tumor development, with implications for deciphering neuroblastoma cell origin. We created an embryonic conditional knockout model and show that cell type-specific loss of MOXD1 leads to disrupted organ homeostasis and failed adrenal gland formation, home for neuroblastoma. We show that MOXD1 is a tumor suppressor gene in zebrafish, chick, and mice in vivo models. One-Sentence SummaryNeural crest-specific MOXD1 is a de novo tumor-suppressor gene in childhood cancers arising during embryogenesis.

cancer biology↗

bioRxiv2021

Automatic end-to-end analysis of high-throughput in vitro cell culture screening by HTSplotter

In biomedical research, high-throughput screening is often applied as it comes with automatization, higher-efficiency, and more and faster results. High-throughput screening experiments encompass drug, drug combination, genetic perturbagen or a combination of genetic and chemical perturbagen screens. These experiments are conducted in real-time assays over time or in an endpoint assay. The data analysis consists of data cleaning and structuring, as well as further data processing and visualisation, which, due to the amount of data, can easily become laborious, time consuming and error-prone. Therefore, several tools have been developed to aid researchers in this process, but these typically focus on specific experimental set-ups and are unable to process data of several time points and genetic-chemical perturbagen screens. To meet these needs, we developed HTSplotter, available as web tool and Python module, which performs automatic data analysis and visualisation of either endpoint or real-time assays from different high-throughput screening experiments: drug, drug combination, genetic perturbagen and genetic-chemical perturbagen screens. HTSplotter implements an algorithm based on conditional statements in order to identify experiment type and controls. After appropriate data normalization, including growth rate normalization, HTSplotter executes downstream analyses such as dose-response relationship and drug synergism assessment by the Bliss independence (BI), Zero Interaction Potency (ZIP) and Highest Single Agent (HAS) methods. All results are exported as a text file and plots are saved in a PDF file. The main advantage of HTSplotter over other available tools is the automatic analysis of genetic-chemical perturbagen screens and real-time assays where growth rate and perturbagen effect results are plotted over time. In conclusion, HTSplotter allows for the automatic end-to-end data processing, analysis and visualisation of various high-throughput in vitro cell culture screens, offering major improvements in terms of versatility, efficiency and time over existing tools.

bioinformatics↗

bioRxiv2021

MYCN-induced nucleolar stress drives an early senescence-like transcriptional program in hTERT-immortalized RPE cells

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as beta-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation. HighlightsO_LIActivation of MYCN attenuates proliferation in RPE1 cells C_LIO_LIGrowth arrest is associated with an early senescence-like transcriptional signature C_LIO_LITranscriptional and phenotypic evidence of nucleolar stress C_LIO_LICDKN1A upregulation in G2 phase primes cells for faltering in subsequent G1 C_LI

cancer biology↗

bioRxiv2020

RRM2 is a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma

Neuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10-15 percent of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease at diagnosis. Despite intensive multi-modal therapies nearly 50 percent of high-risk cases relapse and die of their disease. In contrast to the overall paucity of mutations, high-risk neuroblastoma nearly invariably present with recurrent somatic segmental chromosome copy number variants. For several focal aberrations (e.g. MYCN and LIN28B amplification), the direct role in tumor formation has been established. However, for recurrent aberrations, such as chromosome 2p and 17q gains, the identification of genes contributing to tumor initiation or progression has been challenging due to the scarcity of small segmental gains or amplifications. In this study, we identified and functionally evaluated the ribonucleotide reductase regulatory subunit 2 (RRM2) as a top-ranked 2p putative co-driver and therapeutic target in high-risk neuroblastoma enforcing replicative stress resistance. In vitro knock down and pharmacological RRM2 inhibition highlight RRM2 dependency in neuroblastoma cells, further supported by the finding that co-overexpression of RRM2 in a d{beta}h-MYCN transgenic zebrafish line increased tumor penetrance with 80% and accelerated tumor formation. Given the critical role of RRM2 in replication fork progression and regulation of RRM2 through ATR/CHK1 signaling, we tested combined RRM2 and ATR/CHK1 small molecule inhibition with triapine and BAY1895344/prexasertib respectively, and observed strong synergism, in particular for combined RRM2 and CHK1 inhibition. Transcriptome analysis following combinatorial drugging revealed HEXIM1 as one of the strongest upregulated genes. Using programmable DNA binding of dCas9 with a promiscuous biotin ligase, RRM2 promotor bound proteins were identified including HEXIM1 and NurRD complex members, supporting a cooperative role for HEXIM1 upregulation together with CHK1 inhibition in further attenuating RRM2 expression levels. We evaluated the impact of combined RRM2/CHK1 inhibition in vivo, with treatment of a murine xenograft model showing rapid and complete tumor regression, without tumor regrowth upon treatment arrest. In conclusion, we identified RRM2 as a novel dependency gene in neuroblastoma and promising target for synergistic drug combinations with small compounds targeting DNA checkpoint regulators.

cancer biology↗

bioRxiv2020

Comprehensive benchmarking of single cell RNA sequencing technologies for characterizing cellular perturbation

Technological advances in transcriptome sequencing of single cells continues to provide an unprecedented view on tissue composition and cellular heterogeneity. While several studies have compared different single cell RNA-seq methods with respect to data quality and their ability to distinguish cell subpopulations, none of these studies investigated the heterogeneity of the cellular transcriptional response upon a chemical perturbation. In this study, we evaluated the transcriptional response of NGP neuroblastoma cells upon nutlin-3 treatment using the C1, ddSeq and Chromium single cell systems. These devices and library preparation methods are representative for the wide variety of platforms, ranging from microfluid chips to droplet-based systems and from full transcript sequencing to 3-prime end sequencing. In parallel, we used bulk RNA-seq for molecular characterization of the transcriptional response. Two complementary metrics to evaluate performance were applied: the first is the number and identity of differentially expressed genes as defined in consensus by two statistical models, and the second is the enrichment analysis of biological signals. Where relevant, to make the data more comparable, we downsampled sequencing library size, selected cell subpopulations based on specific RNA abundance features, or created pseudobulk samples. While the C1 detects the highest number of genes per cell and better resembles bulk RNA-seq, the Chromium identifies most differentially expressed genes, albeit still substantially fewer than bulk RNA-seq. Gene set enrichment analyses reveals that detection of a limited set of the most abundant genes in single cell RNA-seq experiments is sufficient for molecular phenotyping. Finally, single cell RNA-seq reveals a heterogeneous response of NGP neuroblastoma cells upon nutlin-3 treatment, revealing putative late-responder or resistant cells, both undetected in bulk RNA-seq experiments.

molecular biology↗

bioRxiv2020

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

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 which is known to induce genomic instability causing 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 versus 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.

cancer biology↗

bioRxiv2020

SOX11 is a lineage-dependency factor and master epigenetic regulator in neuroblastoma

The pediatric extra-cranial tumor neuroblastoma (NB) is characterised by a low mutation burden while copy number alterations are present in most high-risk cases. We identified SOX11 as a strong lineage dependency transcription factor in adrenergic NB based on recurrent chromosome 2p focal gains and amplifications, its specific expression in the normal sympatho-adrenal lineage and adrenergic NBs and its regulation by multiple adrenergic specific cis-interacting (super-)enhancers. Adrenergic NBs are strongly dependent on high SOX11 expression levels for growth and proliferation. Through genome-wide DNA-binding and transcriptome analysis, we identified and validated functional SOX11 target genes, several of which implicated in chromatin remodeling and epigenetic modification. SOX11 controls chromatin accessibility predominantly affecting distal adrenergic lineage-specific enhancers marked by binding sites of the adrenergic core regulatory circuitry. During normal sympathoblast differentiation we find expression of SOX11 prior to members of the adrenergic core regulatory circuitry. Given the broad control of SOX11 of multiple epigenetic regulatory complexes and its presumed pioneer factor function, we propose that adrenergic NB cells have co-opted the normal role of SOX11 as a crucial regulator of chromatin accessibility and cell identity.

cancer biology↗

bioRxiv2019

A comprehensive dataset of TLX1 positive ALL-SIL lymphoblasts and primary T-cell acute lymphoblastic leukemias

Most currently available transcriptome data of T-cell acute lymphoblastic leukemia (T-ALL) are based on polyA[+] RNA sequencing methods thus lacking non-polyadenylated transcripts. Here, we present the data of polyA[+] and total RNA sequencing in the context of in vitro TLX1 knockdown in ALL-SIL cells and a primary T-ALL cohort. We extended this dataset with ATAC sequencing and H3K4me1 and H3K4me3 ChIP sequencing data to map putative gene regulatory regions. In this data descriptor, we present a detailed report of how the data were generated and which bioinformatics analyses were performed. Through several technical validations, we showed that our sequencing data are of high quality and that our in vitro TLX1 knockdown was successful. We also validated the quality of the ATAC and ChIP sequencing data and showed that ATAC and H3K4me3 ChIP peaks are enriched at transcription start sites. We believe that this comprehensive set of sequencing data can be reused by others to further unravel the complex biology of T-ALL in general and TLX1 in particular.

molecular biology↗

bioRxiv2018

SMARTer single cell total RNA sequencing

Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3 end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.

genomics↗

bioRxiv2018

Circulating microRNA biomarkers for metastatic disease in neuroblastoma patients

In this study, the circulating miRNome from diagnostic neuroblastoma serum was assessed for identification of non-invasive biomarkers with potential in monitoring metastatic disease. After determining the circulating neuroblastoma miRNome, 743 miRNAs were screened in two 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 expression 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, suggesting this miRNA signature may be applied to monitor disease burden.

cancer biology↗