Abstract 3545: The Yes-associated protein (YAP) regulates GD2 immunotherapy response in high-risk neuroblastoma
Authors: Pilgrim Adeiye A. , Jonus Hunter C. , Ho Andrew , Cole Anna , Shim Jenny , Goldsmith Kelly C. 
Published: April 2023

Abstract
Background Patients with the pediatric solid tumor high-risk neuroblastoma (HR NB) receive intense multimodal therapy yet 50% still relapse with chemotherapy-resistant disease. Relapsed NBs harbor increased RAS/MAPK pathway mutations and increased expression and downstream activity of the transcriptional co-regulator YAP. We have previously shown that YAP mediates resistance to chemotherapy and MEK inhibitors in RAS mutant NBs (Shim et al., Cancer Res 2020). Patients with relapsed NB are treated with the GD2-targeting monoclonal antibody dinutuximab in combination with chemotherapy. Given the increased expression and activity of YAP in relapsed HR NB, we posited that YAP might also play a role in GD2 immunotherapy response.
Methods/Results We stably knocked down YAP in the human derived NRAS mutant SK-N-AS NB cell line with a scrambled short hairpin (sh) control or 2 YAP-targeting shRNAs to generate 3 distinct cell lines. Dinutuximab requires antibody-dependent cellular cytotoxicity (ADCC) and we have shown that gamma delta (γδ) T cells augment dinutuximab in aggressive NB models. We exposed the shYAP1, shYAP2, and control SK-N-AS cells to γδ T cells with/without dinutuximab. YAP knockdown sensitized both SK-N-AS shYAP cell lines to γδ T cell killing both in the presence and absence of dinutuximab. To investigate the mechanism of increased dinutuximab sensitivity, we evaluated a panel of NB cell lines (MYCN amplified and MYCN single copy) for YAP protein and GD2 cell surface expression and noted an inverse relationship. That same inverse correlation was found for GD2 and YAP gene expression in primary HR NB tumor datasets. We therefore evaluated GD2 expression following YAP knockdown in SK-N-AS and show that GD2 significantly increased on the cell surface following YAP inhibition. PRRX1 is a master transcription factor that induces a mesenchymal NB phenotype which is one of high YAP expression with very low to no GD2 surface expression. Interestingly, PRRX1 expression increased in YAP knockdown cells yet GD2 expression also increased, suggesting YAP regulates GD2 expression more directly than PRRX1. In the GD2 biosynthesis pathway, GM3 is converted into GD3 by GD3 synthase (GD3S). GD2 synthase then catalyzes GD3 into GD2. GD3S gene (ST8SIA1) expression significantly increased (&gt;100-fold) upon YAP knockdown. Furthermore, shRNA stable inhibition of GD3S in shYAP NB cells reverted the phenotype and decreased GD2 cell surface expression back to baseline. We then treated established SK-N-AS control or shYAP xenografts with an 18-day course of human γδ T cells, dinutuximab, and cyclophosphamide. Pilot results show significantly extended survival in mice harboring SK-N-AS shYAP tumors.
Conclusion These results support YAP regulation of GD2 expression through transcriptional suppression of GD3 synthase and identify YAP as a therapeutic target to augment GD2 immunotherapy responses in HR and relapsed NB.
Citation Format: Adeiye A. Pilgrim, Hunter C. Jonus, Andrew Ho, Anna Cole, Jenny Shim, Kelly C. Goldsmith. The Yes-associated protein (YAP) regulates GD2 immunotherapy response in high-risk neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3545.