Inhibition of OCT4 Binding at the MYCN Locus Induces Neuroblastoma Cell Death Accompanied by Downregulation of Transcripts with High-Open Reading Frame Dominance

Abstract

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether other transcription factors contribute to NB cell survival. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN activity. Differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with high-ORF dominance were significantly associated with genes whose high expression is associated with a poor prognosis of NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the human-specific OCT4-MYCN positive feedback loop may serve as an effective therapeutic strategy for MYCN-amplified NB. Contribution to the fieldNeuroblastoma (NB) is a childhood tumor. Amplification of MYCN is frequently observed in high-risk NBs and is linked to a poor prognosis. Multiple transcription factors, including OCT4, MYCN, CTCF, and p53, regulate MYCN expression by binding to the MYCN locus. This study investigated the contribution of these transcription factors in NB cell survival. We used CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in MYCN-amplified NB cell lines. We found that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, reduces MYCN activity and induces NB cell death. A detailed investigation of the molecular mechanisms of cell death revealed that the downregulated transcripts after suppressed MYCN activity were associated with high-open reading frame (ORF) dominance scores, which are associated with translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Reduced expression of these splicing factors altered the PKM mRNA splicing accompanied by the induction of p53-caspase-2-MDM2-mediated cell death. These findings suggest that disrupting the human-specific OCT4-MYCN positive feedback loop may serve as a promising therapeutic strategy for MYCN-amplified NB.