Target In Neuroblastoma Research Articles

Targeting DLK1 in neuroblastoma

An ideal cell surface target has ubiquitously high cancer expression, absence from healthy tissues, and an essential role cancer initiation and/or maintenance. In this issue of Cancer Cell, Hamilton etal. combine proteomics, transcriptomics, epigenomics, and dependency databases to identify DLK1, a novel immunotherapeutic target for neuroblastoma.

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma

Cancer immunotherapies produce remarkable results in B cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor and normal tissues to identify biologically relevant cell surface immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer. Proteogenomic analyses reveal sixty high-confidence candidate immunotherapeutic targets, and we prioritize delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlates with a super-enhancer. Immunofluorescence, flow cytometry, and immunohistochemistry show robust cell surface expression of DLK1. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells results in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Since high DLK1 expression is found in several adult and pediatric cancers, our study demonstrates the utility of a proteogenomic approach and credentials DLK1 as an immunotherapeutic target.

In Silico Investigation of Taurodispacamide A and Strepoxazine A from Agelas oroides S. as Potential Inhibitors of Neuroblastoma Targets Reveals Promising Anticancer Activity

In Silico Investigation of Taurodispacamide A and Strepoxazine A from Agelas oroides S. as Potential Inhibitors of Neuroblastoma Targets Reveals Promising Anticancer Activity

Discovery of organosulfur-based selective HDAC8 inhibitors with anti-neuroblastoma activity

Histone deacetylases (HDACs) are important epigenetic regulators of gene expression and various cellular processes, and are potential targets for anticancer therapy. In particular, HDAC8 is a promising therapeutic target for childhood neuroblastoma. To date, five HDAC inhibitors have been approved as anticancer drugs; however, all are non-selective HDAC inhibitors with various side effects. Furthermore, many promising HDAC inhibitors incorporate hydroxamic acid as a zinc binding group (ZBG), which may be associated with toxicity. Therefore, identification of isoform-selective HDAC inhibitors with novel ZBG is crucial. Here, a series of sulfur-based selective HDAC8 inhibitors featuring a novel ZBG were identified by modifying the early hit, ajoene, a component of garlic. Structure-activity relationship studies uncovered potent and selective HDAC8 inhibitors, and docking studies provided a structural rationale for HDAC8 inhibitory activity. One of the potent compounds, (Z)-1-phenyl-7-(4-methoxyphenyl)-2,3,7-trithiahepta-4-ene-7-oxide (15c), exhibited antiproliferative activity, with a GI50 of 2 µM, against neuroblastoma cell lines. 15c also showed significant in vivo efficacy in a neuroblastoma BE(2)-C xenograft model.

Joint metabolomics and transcriptomics analysis systematically reveal the impact of MYCN in neuroblastoma

The limited understanding of the molecular mechanism underlying MYCN-amplified (MNA) neuroblastoma (NB) has hindered the identification of effective therapeutic targets for MNA NB, contributing to its higher mortality rate compared to MYCN non-amplified (non-MNA) NB. Therefore, a comprehensive analysis integrating metabolomics and transcriptomics was conducted to systematically investigate the MNA NB. Metabolomics analysis utilized plasma samples from 28 MNA NB patients and 68 non-MNA NB patients, while transcriptomics analysis employed tissue samples from 15 MNA NB patients and 37 non-MNA NB patients. Notably, joint metabolomics and transcriptomics analysis was performed. A total of 46 metabolites exhibited alterations, with 21 displaying elevated levels and 25 demonstrating reduced levels in MNA NB. In addition, 884 mRNAs in MNA NB showed significant changes, among which 766 mRNAs were higher and 118 mRNAs were lower. Joint-pathway analysis revealed three aberrant pathways involving glycerolipid metabolism, purine metabolism, and lysine degradation. This study highlights the substantial differences in metabolomics and transcriptomics between MNA NB and non-MNA NB, identifying three abnormal metabolic pathways that may serve as potential targets for understanding the molecular mechanisms underlying MNA NB.

Assessment of Ferroptotic Cell Death and Related Treatment Targets in Neuroblastoma

Assessment of Ferroptotic Cell Death and Related Treatment Targets in Neuroblastoma

Preclinical spheroid models identify BMX as a therapeutic target for metastatic MYCN nonamplified neuroblastoma.

The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.

Molecular profiling of core immune-escape genes highlights TNFAIP3 as an immune-related prognostic biomarker in neuroblastoma.

Neuroblastoma (NB) is the most prevalent and deadliest pediatric solid tumor. With of over 50% of high-risk neuroblastoma cases relapse, the imperative for novel drug targets and therapeutic strategies is accentuated. In neuroblastoma, the existence of tumor-associated macrophages (TAMs) correlates with an unfavorable patient prognosis. However, the clinical relevance and prognostic implications of regulatory genes linked to TAMs infiltration in neuroblastoma remain unclear, and further study is required. We conducted a comprehensive analysis utilizing transcriptome expression profiles from three primary datasets associated with neuroblastoma (GSE45547, GSE49710, TARGET) to identify hub genes implicated in immune evasion within neuroblastoma. Subsequently, we utilized single-cell RNA sequencing analysis on 17 clinical neuroblastoma samples to investigate the expression and distribution of these hub genes, leading to the identification of TNFAIP3. The above three public databases were merged to allowed for the validation of TNFAIP3's molecular functions through GO and KEGG analysis. Furthermore, we assessed TNFAIP3's correlation with immune infiltration and its potential immunotherapeutic impact by multiple algorithms. Our single-cell transcriptome data revealed the role of TNFAIP3 in macrophage polarization. Finally, preliminary experimental verifications to confirm the biological functions of TNFAIP3-mediated TAMs in NB. A total of 6 genes related to immune evasion were screened and we found that TNFAIP3 exhibited notably higher expression in macrophages than other immune cell types, based on the scRNA-sequencing data. GO and KEGG analysis showed that low expression of TNFAIP3 significantly correlated with the activation of multiple oncogenic pathways as well as immune-related pathways. Then validation affirmed that individuals within the TNFAIP3 high-expression cohort could potentially derive greater advantages from immunotherapeutic interventions, alongside exhibiting heightened immune responsiveness. Deciphering the pseudotime trajectory of macrophages, we revealed the potential of TNFAIP3 in inducing the polarization of macrophages towards the M1 phenotype. Finally, we confirmed that patients in the TNFAIP3 high expression group might benefit more from immunotherapy or chemotherapy as substantiated by RT-qPCR and immunofluorescence examinations. Moreover, the role of TNFAIP3 in macrophage polarization was validated. Preliminary experiment showed that TNFAIP3-mediated TAMs inhibit the proliferation, migration and invasion capabilities of NB cells. Our results suggest that TNFAIP3 was first identified as a promising biomarker for immunotherapy and potential molecular target in NB. Besides, the presence of TNFAIP3 within TAMs may offer a novel therapeutic strategy for NB.

Regulatory roles of insulin growth factor binding protein family in neuroblastoma cell proliferation and differentiation: Potential prognostic biomarkers and therapeutic targets for neuroblastoma

AbstractNeuroblastoma (NB), as a representative of tumors of embryonic origin in children, has specific clinical features. On the one hand, a very small number of NBs may appear to regress on their own. On the other hand, highly malignant NBs can invade the surrounding blood vessels and organs and metastasize to distant bone, bone marrow, and lymph nodes in the early stages of the disease. Based on differential affinities to insulin growth factors (IGFs), insulin growth factor binding proteins (IGFBPs) are classified into two groups: IGF binding proteins (IGFBP1‐6) with high‐affinity and IGF low‐affinity binding proteins, such as IGFBP‐related proteins (IGFBP rP1‐10). IGFBP are crucial regulators of the bioavailability and function of IGF in metabolic signaling and as modulators of IGF signaling, and their role in NB is gaining increasing attention. In this study, we investigate the involvement of IGFBP family members in the growth and differentiation of NB cells, as well as the potential of IGFBPs as prognostic biomarkers and therapeutic targets for human NB.

USP44 Overexpression Drives a MYC-Like Gene Expression Program in Neuroblastoma through Epigenetic Reprogramming.

Neuroblastoma is an embryonic cancer that contributes disproportionately to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer, although epigenetic pathways have been implicated in disease pathogenesis. We used an expression-based computational screen that examined the impact of deubiquitinating enzymes on patient survival to identify potential new targets. We identified the histone H2B deubiquitinating enzyme USP44 as the enzyme with the greatest impact on survival in patients with neuroblastoma. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN amplification. The subset of patients with tumors expressing high levels of USP44 had significantly worse survival, including those with tumors lacking MYCN amplification. We showed experimentally that USP44 regulates neuroblastoma cell proliferation, migration, invasion, and neuronal development. Depletion of the histone H2B ubiquitin ligase subunit RNF20 resulted in similar findings, strongly implicating this histone mark as the target of USP44 activity in this disease. Integration of transcriptome and epigenome in analyses demonstrates a distinct set of genes that are regulated by USP44, including those in Hallmark MYC target genes in both murine embryonic fibroblasts and the SH-SY5Y neuroblastoma cell line. We conclude that USP44 is a novel epigenetic regulator that promotes aggressive features and may be a novel target in neuroblastoma. Implications: This study identifies a new genetic marker of aggressive neuroblastoma and identifies the mechanisms by which its overactivity contributes to the pathophysiology of this disease.

EGCG Disrupts the LIN28B/Let-7 Interaction and Reduces Neuroblastoma Aggressiveness.

Neuroblastoma (NB) is the most commonly diagnosed extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with a high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and the identification of novel potential therapeutic targets and agents is an urgent clinical need. The RNA-binding protein LIN28B has been identified as an oncogene in NB and is associated with a poor prognosis. Given that LIN28B acts by negatively regulating the biogenesis of the tumor suppressor let-7 miRNAs, we reasoned that selective interference with the LIN28B/let-7 miRNA interaction would increase let-7 miRNA levels, ultimately leading to reduced NB aggressiveness. Here, we selected (-)-epigallocatechin 3-gallate (EGCG) out of 4959 molecules screened as the molecule with the best inhibitory activity on LIN28B/let-7 miRNA interaction and showed that treatment with PLC/PLGA-PEG nanoparticles containing EGCG (EGCG-NPs) led to an increase in mature let-7 miRNAs and a consequent inhibition of NB cell growth. In addition, EGCG-NP pretreatment reduced the tumorigenic potential of NB cells in vivo. These experiments suggest that the LIN28B/let-7 miRNA axis is a good therapeutic target in NB and that EGCG, which can interfere with this interaction, deserves further preclinical evaluation.

The MYCN 5′ UTR as a therapeutic target in neuroblastoma

Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.

Choosing T-cell sources determines CAR-T cell activity in neuroblastoma.

The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.

Abstract 5988: The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma

Abstract Dysregulation of the Ubiquitin Proteasome System has been linked to many human diseases, including cancer. Expression of UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes and its functional roles in neuroblastoma pathogenesis are not known. We have recently identified a ubiquitin complex ITCH/UBE4B which mediates ubiquitination of Ku70 and c-FLIPL proteins for proteasomal degradation allowing HDAC inhibitor-mediated caspase-8 dependent apoptosis. We also confirmed that the deubiquitinase USP7, critical player in tumor suppression and DNA repair, can be a potential therapeutic target for neuroblastoma. Highly selective USP7 inhibitors have demonstrated significant antitumor activity in preclinical models of adult cancer, and we reported that these inhibitors alone can be more effective against neuroblastoma tumor growth. Our hypothesis is that USP7 inhibition will be more effective against neuroblastoma tumors through destabilization of ITCH/UBE4B complex protein targets allowing a stronger induction of cell death in response to treatment for the children. To evaluate efficacy of USP7 inhibitors in combination with HDAC inhibitors or chemotherapy against neuroblastoma tumor growth, neuroblastoma cell lines depleted for UBE4B or USP7 were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and apoptosis by caspase cleavage and PARP cleavage detection by western blot. We have evaluated also, whether ubiquitination/deubiquitination and degradation rates of p53, Ku70 and c-FLIPL proteins could modulate induction of apoptosis and necroptosis. USP7 inhibition resulted in decreases in cell viability in p53 wild-type neuroblastoma cell lines. USP7 inhibition induced apoptosis and necroptosis by increasing phosphorylation of MLKL, RIPK3 and RIPK1. In UBE4B or USP7 depleted cells, a huge induction of necroptosis and a small rate of apoptosis were observed. We also identified USP7 as a deubiquitinase of Ku70, stabilizing Ku70 at the basal level, leading to stabilization of Ku70/c-FLIPL/Bax complex. USP7 inhibition destabilizes ku70 and c-FLIPL for protein degradation leading to induction of apoptosis as well as necroptosis. UBE4B depletion in neuroblastoma inhibits HDAC inhibitors mediated caspase-8 mediated apoptosis but enhances necroptosis, due to the inhibition of Ku70 and c-FLIPL proteins degradation leading to the blockade of caspase-8 activation and the activation of the necroptosome. Our data suggests that USP7 and ITCH/UBE4B can control the switch between apoptosis and necroptosis in response to USP7 and HDAC inhibitors. USP7 inhibition and ITCH/UBE4B activation by HDAC inhibitors could be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, CARLA SAMPAIO, PETER E. ZAGE. The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5988.

Potential pharmacological mechanisms of tanshinone IIA in the treatment of human neuroblastoma based on network pharmacological and molecular docking Technology.

Tanshinone IIA (Tan-IIA) is the main bioactive component of Chinese herbal medicine salvia miltiorrhiza (Danshen). Sodium sulfonate of Tan-IIA is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tan-IIA also has inhibitory effects on tumor cells such as gastric cancer, but its therapeutic effect and mechanism on human neuroblastoma have not been evaluated, so its pharmacological mechanism is systematically evaluated by the combined method of network pharmacology and molecular docking. PharmMapper and SwissTargetPrediction predicted 331 potential Tan-IIA-related targets, and 1,152 potential neuroblastoma-related targets were obtained from GeneCards, DisGeNET, DrugBank, OMIM and Therapeutic Target databases (TTD), 107 common targets for Tan-IIA and neuroblastoma. Through gene ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomesa (KEGG) pathway enrichment, protein-protein interaction (PPI) network and cytoHubba plug-in, 10 related signal pathways (Pathways in cancer, PI3K-Akt signaling pathway, Prostate cancer, etc.) and 10 hub genes were identified. The results of molecular docking showed that Tan-IIA could interact with 10 targets: GRB2, SRC, EGFR, PTPN1, ESR1, IGF1, MAPK1, PIK3R1, AKT1 and IGF1R. This study analyzed the related pathways and targets of Tan-IIA in the treatment of human neuroblastoma, as well as the potential anticancer and anti-tumor targets and related signaling pathways of Tan-IIA, which provides a reference for us to find and explore effective drugs for the treatment of human neuroblastoma.

AF1q is a universal marker of neuroblastoma that sustains N-Myc expression and drives tumorigenesis

Neuroblastoma is the most common extracranial malignant tumor of childhood, accounting for 15% of all pediatric cancer deaths. Despite significant advances in our understanding of neuroblastoma biology, five-year survival rates for high-risk disease remain less than 50%, highlighting the importance of identifying novel therapeutic targets to combat the disease. MYCN amplification is the most frequent and predictive molecular aberration correlating with poor outcome in neuroblastoma. N-Myc is a short-lived protein primarily due to its rapid proteasomal degradation, a potentially exploitable vulnerability in neuroblastoma. AF1q is an oncoprotein with established roles in leukemia and solid tumor progression. It is normally expressed in brain and sympathetic neurons and has been postulated to play a part in neural differentiation. However, no role for AF1q in tumors of neural origin has been reported. In this study, we found AF1q to be a universal marker of neuroblastoma tumors. Silencing AF1q in neuroblastoma cells caused proteasomal degradation of N-Myc through Ras/ERK and AKT/GSK3β pathways, activated p53 and blocked cell cycle progression, culminating in cell death via the intrinsic apoptotic pathway. Moreover, silencing AF1q attenuated neuroblastoma tumorigenicity in vivo signifying AF1q’s importance in neuroblastoma oncogenesis. Our findings reveal AF1q to be a novel regulator of N-Myc and potential therapeutic target in neuroblastoma.

MTHFD1 regulates the NADPH redox homeostasis in MYCN-amplified neuroblastoma.

MYCN amplification is an independent poor prognostic factor in patients with high-risk neuroblastoma (NB). Further exploring the molecular regulatory mechanisms in MYCN-amplified NB will help to develop novel therapy targets. In this study, methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) was identified as the differentially expressed gene (DEG) highly expressed in MYCN-amplified NB, and it showed a positive correlation with MYCN and was associated with a poor prognosis of NB patients. Knockdown of MTHFD1 inhibited proliferation and migration, and induced apoptosis of NB cells in vitro. Mouse model experiments validated the tumorigenic effect of MTHFD1 in NB in vivo. In terms of the mechanism, ChIP-qPCR and dual-luciferase reporter assays demonstrated that MTHFD1 was directly activated by MYCN at the transcriptional level. As an important enzyme in the folic acid metabolism pathway, MTHFD1 maintained the NADPH redox homeostasis in MYCN-amplified NB. Knockdown of MTHFD1 reduced cellular NADPH/NADP+ and GSH/GSSG ratios, increased cellular reactive oxygen species (ROS) and triggered the apoptosis of NB cells. Moreover, genetic knockdown of MTHFD1 or application of the anti-folic acid metabolism drug methotrexate (MTX) potentiated the anti-tumor effect of JQ1 both in vitro and in vivo. Taken together, MTHFD1 as an oncogene is a potential therapeutic target for MYCN-amplified NB. The combination of MTX with JQ1 is of important clinical translational significance for the treatment of patients with MYCN-amplified NB.

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.

METTL3 drives telomere targeting of TERRA lncRNA through m6A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma.

Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+NB.

Investigation of differentially expressed genes related to cellular senescence between high-risk and non-high-risk groups in neuroblastoma.

This study aims to identify differentially expressed genes (DEGs) between high-risk and non-high-risk groups in neuroblastoma (NB), construct a prognostic model, and establish a risk score formula. The NB dataset GSE49710 (n = 498) from the GEO database served as the training cohort to select DEGs between high-risk and non-high-risk NB groups. Cellular senescence-related genes were obtained from the Aging Atlas database. Intersection genes from both datasets were identified as key genes of cellular senescence-related genes (SRGs). A prognostic model was constructed using Univariate Cox regression analysis and the Lasso algorithm with SRGs. Validation was performed using the E-MTAB-8248 cohort (n = 223). The expression levels of AURKA and CENPA were evaluated via RT-qPCR in two clinical NB sample groups. Eight SRGs were identified, and a prognostic model comprising five genes related to cellular senescence was constructed. AURKA and CENPA showed significant expression in clinical samples and were closely associated with cellular senescence. The prognostic model consisted with five cellular senescence related genes effectively predicts the prognosis of NB patients. AURKA and CENPA represent promising targets in NB for predicting cellular senescence, offering potential insights for NB therapy.