Aida Amador-Álvarez

Neuroblastoma is a neural crest-derived pediatric malignancy characterized by marked cellular heterogeneity and variable differentiation status. Undifferentiated tumors are associated with aggressive clinical behavior, treatment resistance and poor outcome, highlighting the need to identify molecular mechanisms that sustain tumor cell plasticity and prevent differentiation. Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase involved in cell-cycle progression, DNA-damage responses and transcriptional regulation, and has previously been associated with neuroblastoma progression. However, its role in the control of neuroblastoma differentiation remains unclear. Here, we investigated the relationship between VRK1 expression, tumor differentiation and stem-like properties in human neuroblastoma. Analysis of patient tumor datasets and tissue microarrays showed that VRK1 expression is enriched in undifferentiated neuroblastoma and stage 4 tumors, and inversely correlates with established differentiation markers, including DDC, NCAM1 and S100B. This association was maintained in MYCN-non-amplified tumors, indicating that the relationship between VRK1 and differentiation is not dependent on MYCN status. Single-cell transcriptomic analyses further demonstrated elevated VRK1 expression in developmentally immature neural crest progenitor and Schwann cell precursor-like populations. Induction of neuronal or mesenchymal differentiation consistently reduced VRK1 expression in neuroblastoma cell lines and patient-derived cells. Conversely, VRK1 silencing promoted differentiation-marker expression, reduced nestin and Ki67 expression, and produced sustained differentiation-associated changes in xenograft tumors. VRK1 was also enriched in tumorsphere cultures that select for undifferentiated stem-like neuroblastoma cells. VRK1 depletion impaired tumorsphere growth, reduced intratumoral proliferation and altered the balance between undifferentiated cells and differentiated progeny, supporting a role for VRK1 in self-renewal and maintenance of progenitor-like tumor cells. Mechanistically, VRK1 expression positively correlated with the core stemness transcription factor SOX2 in neuroblastoma tumor cells. VRK1 knockdown reduced nuclear SOX2 abundance, whereas VRK1 overexpression increased SOX2 protein levels. In addition, analysis of the VRK1 locus identified an active chromatin configuration and potential SOX2-binding sites, consistent with a regulatory relationship between these factors. Together, these findings identify VRK1 as a regulator of the undifferentiated, proliferative and stem-like state in neuroblastoma. The VRK1-SOX2 axis may contribute to stabilizing tumor-cell immaturity and represents a potential target for differentiation based therapeutic strategies in high-risk neuroblastoma.

Background: Neuroblastoma (NB) is a pediatric cancer with highly variable outcomes, necessitating improved understanding of the molecular pathways driving its progression. Intratumor cellular heterogeneity related to neural differentiation has emerged as a defining characteristic that can explain its aggressive behavior. Although recurrent driver mutations are not typically observed in these tumors, Rho GTPases signaling genes have been identified as one of the most frequently mutated in aggressive NB cases. Rho GTPases are key regulators of cell morphology, migration, and differentiation, yet their role in NB remains underexplored. This study aims to comprehensively evaluate the expression and clinical significance of Rho GTPase signaling networks in NB tumors. Methods: We analyzed the expression profiles of Rho GTPases, their regulators, and effectors, across multiple NB patient cohorts. Gene expression correlations with clinical parameters were assessed, and bioinformatics analyses were employed to identify gene expression patterns and interactions in tumors. Functional studies were performed in NB cell lines and in vivo models to validate the role of key Rho GTPases, including Cdc42, in NB progression and differentiation. Results: Our analysis revealed widespread dysregulation of Rho GTPase signaling in NB tumors. Specific GTPases, such as RHOA or RHOV , were upregulated in advanced disease stages, while others, including RHOB , RHOU and CDC42 , were downregulated and associated with poor prognosis. A minimal Rho-related gene signature was identified as a strong predictor of NB patient survival. Functional validation highlighted Cdc42 as a key regulator of NB differentiation, where its downregulation was necessary for maintaining the malignant, undifferentiated phenotype of NB cells. We also identified ARHGAP31 /CdGAP as a critical regulator of Cdc42 in NB progenitor cells, suggesting a mechanism for Cdc42 suppression in aggressive NB. Conclusions: An important role for Rho GTPase signaling in NB progression is revealed, providing a foundation for further exploration of Rho GTPase-targeted therapies in NB. In particular, Cdc42 signaling intervene in the balance between differentiation and stemness in NB cells, suggesting specific signaling events controlling the identity and plasticity of NB cells.

Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology.

Neuroblastoma (NB) is one of the most common pediatric cancers and presents a poor survival rate in affected children. Current pretreatment risk assessment relies on a few known molecular parameters, like the amplification of the oncogene MYCN. However, a better molecular knowledge about the aggressive progression of the disease is needed to provide new therapeutical targets and prognostic markers and to improve patients’ outcomes. The human protein kinase VRK1 phosphorylates various signaling molecules and transcription factors to regulate cell cycle progression and other processes in physiological and pathological situations. Using neuroblastoma tumor expression data, tissue microarrays from fresh human samples and patient-derived xenografts (PDXs), we have determined that VRK1 kinase expression stratifies patients according to tumor aggressiveness and survival, allowing the identification of patients with worse outcome among intermediate risk. VRK1 associates with cell cycle signaling pathways in NB and its downregulation abrogates cell proliferation in vitro and in vivo. Through the analysis of ChIP-seq and methylation data from NB tumors, we show that VRK1 is a MYCN gene target, however VRK1 correlates with NB aggressiveness independently of MYCN gene amplification, synergizing with the oncogene to drive NB progression. Our study also suggests that VRK1 inhibition may constitute a novel cell-cycle-targeted strategy for anticancer therapy in neuroblastoma.

The luxCDABE operon of Photorhabdus luminescens can be used as a bioluminescent reporter to measure gene transcription nondestructively. Here we describe protocols to (1) generate random transcriptional fusions of the lux operon to genes of the Salmonella genome, (2) screen for specific fusions with constitutive expression, Salmonella pathogenicity island 1-related expression, or Salmonella pathogenicity island 2-related expression, and (3) determine the site of luxCDABE integration.