METTL3/MYCN cooperation drives neural crest differentiation and provides therapeutic vulnerability in neuroblastoma

Neuroblastoma (NB) is a childhood cancer. Improper differentiation of developing trunk neural crest cells (tNCC) cause NB tumor formation in sympathetic nervous system. N6-methyladenosine (m6A) epitranscriptomic modification control post-transcriptional gene expression but the mechanism which recruit m6A methyltransferase complex to specific locus is less characterized. We explored if m6A epitranscriptome could fine tune gene regulation in migrating/differentiating tNCC. We uncovered m6A epitranscriptome based regulation of HOX gene expression in tNCC which contributes in their timely differentiation to sympathetic neurons. Posterior HOX genes are m6A modified in MYCN NB cells and tumors. We provided evidence that sustained overexpression of MYCN oncogene could drive METTL3 recruitment in subset of genes to create an undifferentiated state in tNCC. Our study suggests oncogenic transcription factor mediated modulation of m6A epitranscriptome could be a prevalent mechanism in other cancers. METTL3 depletion/inhibition induces DNA damage and differentiation in MYCN overexpressed cells. METTL3 inhibition increases vulnerability to the chemotherapeutic drug in MYCN-amplified Patient-derived xenografts (PDX) cells, suggesting METTL3 inhibition could be a potential therapeutic approach for NB. RNA-seq was performed in bilogical replicates from total RNA isolated at different stages of hESC differentiation to Sympathtic Neurons (SN) or neural crest stem cells NCSC. RNA from MYCN amplified NB cell line (SKNBE2) and tumor samples were also sequenced. RNA-seq was also performed in cells with METTL3 KD and with induction of MYCN overexpression. m6A- RNA immunoprecipitaion followed by sequencing (m6A RIP-seq) was performed with RNA isolated fromf hESC, trunk neural crest cells (tNCC), neural crest stem cells (NCSC), MYCN amplified NB cell line (SKNBE2) and tumor samples. Chromatin immunoprecipitaion followed by sequencing for METTL3, MYCN and H3K27ac in hESC and tNCC. In SHEP cells with or without induction of MYCN overexpression ChIP-seq was performed with METTL3 and MYCN antibodies. All the ChIP were performed in duplicates and pooled before library prepation step.