# YAP1 Enhances Mesenchymal-Type Gene Expression in Human Adrenergic-Type Neuroblastoma Cells

## TL;DR

This study shows that the YAP1 protein helps neuroblastoma cells become more resistant to treatment by shifting to a mesenchymal state, which could lead to new therapies.

## Contribution

The study identifies YAP1 as a driver of mesenchymal transformation and chemotherapy resistance in neuroblastoma cells.

## Key findings

- YAP1 activates mesenchymal gene expression in neuroblastoma cell models.
- YAP1 activity overlaps with genes associated with poor prognosis in patient tumors.
- Constitutive YAP1 activation makes neuroblastoma cells resistant to chemotherapy.

## Abstract

More than half of infants and children with high-risk neuroblastoma will experience relapse despite intensive therapy that includes multiple chemotherapy agents. Survival is as low as 20% after relapse. To improve survival, we must better understand why these tumors are so resistant to treatment, often even before prior exposure to chemotherapy. Molecular studies of relapsed tumors indicate that neuroblastoma cells are capable of varying their nature and capabilities without altering the genome. The mesenchymal cell form was strongly enriched in relapsed tumors. We show here that the YAP1 protein plays a role in shifting the neuroblastoma cell state towards the more difficult-to-treat mesenchymal form. We performed functional experiments and identified a profile of genes active when YAP1 is acting in neuroblastoma cell models and confirmed this profile in patient tumor samples. Our results help to better understand a facet of high-risk neuroblastoma that helps evade chemotherapy.

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 YAP1S127A. 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 YAP1S127A 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, YAP1S127A 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.