NeuroD1-USP1-MYCN axis drives tumor progression in neuroblastoma

Neuroblastoma, originating from the sympathetic neural crest, is the most prevalent extracranial solid tumor in children. Amplification of MYCN is a widely recognized indicator of poor prognosis in neuroblastoma. However, the structural properties of the N-Myc protein encoded by MYCN have impeded the development of direct inhibitors with favorable drug-like properties. This study aimed to investigate the upstream regulatory mechanisms of N-Myc stabilization in neuroblastoma and explore potential therapeutic strategies targeting these mechanisms.

The regulatory role of NeuroD1 in neuroblastoma was evaluated through in vitro and in vivo experiments. Mechanistic studies were performed to examine the effects of NeuroD1 knockdown on N-Myc ubiquitination and degradation. Transcriptional target screening through RNAseq and ChIPseq was conducted to identify downstream effectors of NeuroD1, and the interaction between USP1 and N-Myc was assessed by co-IP and western blot. The therapeutic efficacy of Pimozide was investigated in neuroblastoma cells in vitro.

NeuroD1 was identified as a critical regulator associated with MYCN amplification. NeuroD1 promoted the proliferation of neuroblastoma cells in vitro and in vivo. Mechanistically, NeuroD1 knockdown increased K48-linked polyubiquitination of N-Myc, leading to its proteasomal degradation. USP1 was identified as a key downstream effector of NeuroD1 and was shown to interact with N-Myc, removing K48-linked polyubiquitin chains and stabilizing the protein. Pimozide effectively suppressed USP1 expression, reduced N-Myc levels, and inhibited neuroblastoma cell proliferation.

This study uncovered a novel oncogenic axis in neuroblastoma, where NeuroD1 transcriptionally upregulates USP1, promoting N-Myc stabilization and tumor progression. Furthermore, the findings highlight the therapeutic potential of repurposing Pimozide as a promising treatment strategy for this aggressive tumor subtype.