For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our “neuroblastoma hybrid capture sequencing panel” (NB-HCSP) assay employs a technology for the high-coverage sequencing (>1000×) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection.

Background: The KDM1A histone demethylase regulates the cellular balance between proliferation and differentiation, and is often deregulated in human cancers including the childhood tumor neuroblastoma. We previously showed that KDM1A is strongly expressed in undifferentiated neuroblastomas and correlates with poor patient prognosis, suggesting a possible clinical benefit from targeting KDM1A. Methods: Here, we tested the efficacy of NCL-1, a small molecule specifically inhibiting KDM1A in preclinical models for neuroblastoma. Results: NCL-1 mimicked the effects of siRNA-mediated KDM1A knockdown and effectively inhibited KDM1A activity in four neuroblastoma cell lines and a patient-representative cell model. KDM1A inhibition shifted the aggressive tumor cell phenotypes towards less aggressive phenotypes. The proliferation and cell viability was reduced, accompanied by the induction of markers of neuronal differentiation. Interventional NCL-1 treatment of nude mice harboring established neuroblastoma xenograft tumors reduced tumor growth and inhibited cell proliferation. Reduced vessel density and defects in blood vessel construction also resulted, and NCL-1 inhibited the growth and tube formation of HUVEC-C cells in vitro. Conclusions: Inhibiting KDM1A could attack aggressive neuroblastomas two-fold, by re-directing tumor cells toward a less aggressive, slower-growing phenotype and by preventing or reducing the vascular support of large tumors.

Background/Objectives: Neuroblastoma cells are phenotypically plastic, transitioning between mesenchymal and adrenergic states. Core functional genes (e.g., YAP1) mark the mesenchymal state, which is linked to unfavorable prognosis. We and others previously demonstrated relapse-specific Hippo-YAP pathway activation in matched primary/relapsed neuroblastomas. Here we explored the role of YAP1 in neuroblastoma aggressiveness and response to therapy. Methods: RT-qPCR and immunoblotting assessed YAP1 expression in neuroblastoma cell lines. RNA-sequencing detected YAP1-dependent gene signatures in Tet-ON SK-N-AS and SH-EP neuroblastoma cell models expressing wildtype YAP1 or constitutively activated YAP1^S127A. Data from cell models were compared with our published YAP1 expression data from neuroblastomas. Efficacy of commonly used chemotherapeutics was comparatively analyzed in the cell models. Results: YAP1 expression showed marked variability across a panel of neuroblastoma cell lines, assessed by mRNA analysis in 10 cell lines and protein analysis in a subset of 9 cell lines. RNA sequencing in constitutively activated YAP1^S127A mutant and wildtype YAP1 models detected 2162 and 1837 significantly differentially expressed genes in the SK-N-AS and SH-EP backgrounds, respectively. Continuously activating YAP1 in SK-N-AS cells upregulated mesenchymal signature genes and mesenchymal-associated transcription factors. Gene expression influenced by YAP1 activity in the cell models significantly overlapped with YAP1-associated genes (e.g., CYR61 and SPRY4) in published tumor data. Functionally, YAP1^S127A expression rendered neuroblastoma cells resistant to chemotherapy. Conclusions: Findings corroborate the idea of a mechanistic role for YAP1 in neuroblastoma adrenergic to mesenchymal reprogramming and therapy resistance. The YAP1-mediated plastic switch towards a mesenchymal expression state in neuroblastoma cells may provide the molecular basis for novel therapeutic avenues.