Transcriptional regulation of genes by MYCN in PAX3::FOXO1-positive rhabdomyosarcomas and their roles in cell cycle progression

Abstract

Background

MYCN amplification and high expression is associated with pediatric malignancies including neuroblastoma and alveolar rhabdomyosarcoma. MYCN transcription in alveolar rhabdomyosarcomas is driven by a feedback loop with the PAX3::FOXO1 fusion protein. However, the role of MYCN is not well-defined.

Methods

Chromatin ImmunoPrecipitation (ChIP)-sequencing of alveolar rhabdomyosarcoma cell lines was used to identify genome-wide MYCN binding sites. Ontology analyses of genes adjacent to MYCN binding sites corresponding to expression changes after siRNA reduction was performed and confirmed by ChIP-qPCR. Cells from each phase of the cell cycle were isolated by Fluorescence Activated Cell Sorting for assessing protein expression by Western blotting.

Results

Genes encoding transcription factors adjacent to MYCN binding sites and genes with binding sites proximal to their promoters were strongly associated with RNA synthesis and cell cycle pathways, respectively. MYCN binding sites in regions that positively correlated with gene expression changes were linked to cell cycle regulation, consistent with phenotypic absence of cell cycle checkpoint control. Cell cycle regulated genes CDK4 and KDM4B were validated as MYCN-regulated and, in-keeping with unchecked cell cycle progression, expressed throughout the cell cycle, coincident with MYCN.

Conclusions

MYCN binding sites associated with gene expression changes defined the contribution of MYCN to the transcriptional control of key pathways/molecular processes in the development and progression of rhabdomyosarcomas. MYCN aberrantly regulated CDK4 and KDM4B expression throughout the cell cycle in PAX3::FOXO1 positive rhabdomyosarcoma. The regulatory network defined supports MYCN, CDK4 and KDM4B as therapeutic targets in the treatment of rhabdomyosarcoma patients.