De Novo Regulation of RD3 Synthesis in Residual Neuroblastoma Cells After Intensive Multi-modal Clinical Therapy Harmonizes Disease Evolution

Most high-risk neuroblastomas that initially respond to therapy will ultimately relapse. Currently, no curative treatment is available. Acquired genetic/molecular rearrangement in therapy-resistant cells contributes to tumor relapse. Recently, we identified significant RD3 loss in progressive disease (PD) and defined its association with advanced disease-stage and poor clinical outcomes. Here, we investigated whether RD3 loss is an acquired process in cells that survive intensive multi-modal clinical therapy (IMCT) and its significance in disease evolution. RD3 status (mRNA, protein) during diagnosis (Dx) and PD after IMCT was investigated in NB patient cohort (n = 106), stage-4 NB cell lines (n = 15) with known treatment status and validated with independent data from another set of 15 cell-lines. Loss of RD3 in metastatic disease was examined using a mouse model of PD and metastatic-site-derived aggressive cells (MSDACs) ex vivo. RD3 silencing/expression assessed changes in metastatic state. Influence of RD3 loss in therapy resistance was examined through independent in vitro and in vivo studies. A significant loss of RD3 mRNA and protein was observed in resistant cells derived from patients with PD after IMCT. This is true to the effect within and between patients. Results from the mouse model identified significant transcriptional/translational loss of RD3 in metastatic tumors and MSDACs. RD3 re-expression in MSDACs and silencing RD3 in parental cells defined the functional relevance of RD3-loss in PD pathogenesis. Analysis of independent studies with salvage therapeutic agents affirmed RD3 loss in surviving resistant cells and residual tumors. The profound reductions in RD3 transcription indicate the de novo regulation of RD3 synthesis in resistant cells after IMCT. Defining RD3 loss in PD and the benefit of targeted reinforcement could improve salvage therapy for progressive neuroblastoma.