A Dual High-Throughput Approach to Enhance Drug Repurposing in Neuroblastoma: Boosting GD2 Expression and Reducing Immune Checkpoint Molecules

Abstract

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 8-10% of all pediatric cancers. Despite the increase in survival of high-risk neuroblastoma patients following the implementation of Dinutuximab, an anti-GD2 monoclonal antibody, into the standard of care, ~50% of these patients relapse, resulting in a poor chance of survival. One of the causes of relapse following immunotherapy can be attributed to the heterogeneous expression of GD2 and the upregulation of immune checkpoint molecules that drive the immunosuppressive tumor microenvironment of neuroblastoma. Tazemetostat has been previously used to upregulate GD2; however, the study failed to address non-responder cell lines. This study aims to repurpose compounds from the standard compound library from Princess Maxìma Centre to find compounds that can upregulate GD2 and downregulate immune checkpoint molecules. Here, we present a protocol for high-throughput drug screening with a high-throughput flow cytometry readout. Our findings show several compound hits that can upregulate GD2, such as Pyrvinium, Sunitinib, and Linsitinib, and compounds that downregulate immune checkpoint molecules, such as Nilotinib, Selumetinib, and S63845. There is no known direct link between the compound treatment and the upregulation or downregulation of markers, and more validation assays are required to further validate their use in combination with Dinutuximab. These compounds open new prospects for the regulation of GD2 and ICs and the future prevention of relapses following immunotherapy to increase the efficacy of high-risk neuroblastoma treatment