MYCN-induced nucleolar stress drives an early senescence-like transcriptional program in hTERT-immortalized RPE cells

Sofia Zanotti,Jolien Van Laere,Michael D Hogarty,Dieter Deforce,Winnok H De Vos,Liselot M Mus,Laurentijn Tilleman,Filip Van Nieuwerburgh,Joni Van Der Meulen,Kaat Durinck,Frank Speleman,Marlies Verschuuren,Suzanne Vanhauwaert,Bieke Decaesteker,Volodimir Olexiouk,Christophe Van Neste

doi:10.1038/s41598-021-93863-9

Abstract

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as β-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation.

Highlights

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma
In contrast with the known growth promoting effect of MYCN, we observed attenuated cell growth accompanied with nucleolar coalescence, appearance of cytoplasmic granules and increased p21 and p53 protein levels
Bulk RNA sequencing was performed at three sequential time points post induction (p.i.) (24 h, 48 h, 72 h) for MYCN-ON and 4-OH tamoxifen (4-OHT)-treated wild type RPE1 (WT) cells, as well as MYCN-OFF and mocktreated WT cells (0 h) (Supplementary Table 1)

Summary

Introduction

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. While mutational burden in these tumors is low, MYCN amplified neuroblastomas and medulloblastoma often exhibit large copy number alterations, most notably 17q gain[2,3], indicating that additional gene dosage effects are required to support tumor initiation and/or progression Given their structural properties, the MYC proteins have been considered as undruggable, but new insights into their function and protein interactions are providing exciting insights towards novel strategies to target these oncogenes. Based on protein-interactions and further in-depth biochemical studies by the Eilers team[1], MYC(N) oncogenicity was attributed to its role in enhancing transcription-stress resilience in tumour cells This includes roles in alleviating replication fork stalling resulting from nucleotide depletion and POL II stalling, R-loop formation and replication-transcription conflicts[5]. We further found that MYCN-induced growth deceleration is accompanied with a transcriptional signature that resembles early senescence induction offering a platform for further mechanistic studies to explore cellular processes that protect against early MYCN-induced cellular transformation

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Conclusion