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Neuroblastoma, a rare embryonic tumor arising from neural crest development, is responsible for 15% of pediatric cancer-related deaths. Recently, several single-cell transcriptome studies were performed on neuroblastoma patient samples to investigate the cell of origin and tumor heterogeneity. However, these individual studies involved a small number of tumors and cells, limiting the conclusions that could be drawn. To overcome this limitation, we integrated seven single-cell or single-nucleus datasets into a harmonized cell atlas covering 362,991 cells across 61 patients. We use this atlas to decipher the transcriptional landscape of neuroblastoma at single-cell resolution, revealing associations between transcriptomic profiles and clinical outcomes within the tumor compartment. In addition, we characterize the complex immune-cell landscape and uncover considerable heterogeneity among tumor-associated macrophages. Finally, we showcase the utility of our atlas as a resource by expanding it with additional data and using it as a reference for data-driven cell-type annotation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Cancer.)

Studies defining normal and disrupted human neural crest cell development have been challenging given its early timing and intricacy of development. Consequently, insight into the early disruptive events causing a neural crest related disease such as pediatric cancer neuroblastoma is limited. To overcome this problem, we developed an in vitro differentiation model to recapitulate the normal in vivo developmental process of the sympathoadrenal lineage which gives rise to neuroblastoma. We used human in vitro pluripotent stem cells and single-cell RNA sequencing to recapitulate the molecular events during sympathoadrenal development. We provide a detailed map of dynamically regulated transcriptomes during sympathoblast formation and illustrate the power of this model to study early events of the development of human neuroblastoma, identifying a distinct subpopulation of cell marked by SOX2 expression in developing sympathoblast obtained from patient derived iPSC cells harboring a germline activating mutation in the anaplastic lymphoma kinase (ALK) gene., Competing Interests: A.K. is a consultant to Novartis, Rgenta, and Blueprint Medicines. L.S. is a scientific co-founder and consultant of Bluerock Therapeutics Inc. All other authors declare no conflict of interest., (© 2023 The Author(s).)

The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC., (© 2023. The Author(s).)

Aurora kinase A (AURKA) is a well-established target in neuroblastoma (NB) due to both its catalytic functions during mitosis and its kinase-independent functions, including stabilization of the key oncoprotein MYCN. We present a structure-activity relationship (SAR) study of MK-5108-derived PROTACs against AURKA by exploring different linker lengths and exit vectors on the thalidomide moiety. PROTAC SK2188 induces the most potent AURKA degradation (DC 50,24h 3.9 nM, D max,24h 89%) and shows an excellent binding and degradation selectivity profile. Treatment of NGP neuroblastoma cells with SK2188 induced concomitant MYCN degradation, high replication stress/DNA damage levels and apoptosis. Moreover, SK2188 significantly outperforms the parent inhibitor MK-5108 in a cell proliferation screen and patient-derived organoids. Furthermore, altering the attachment point of the PEG linker to the 5-position of thalidomide allowed us to identify a potent AURKA degrader with a linker as short as 2 PEG units. With this, our SAR-study provides interesting lead structures for further optimization and validation of AURKA degradation as a potential therapeutic strategy in neuroblastoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.