The roles of RNA m6A modification in disease mechanisms of high-risk neuroblastoma

Abstract

Epitranscriptomics involves covalent modifications of RNA bases, with N6-methyladenosine (m6A) being one of the most abundant and functionally characterized. This modification plays a key role in RNA metabolism, cellular differentiation, and DNA damage response. Although m6A has been implicated in various cancers, its role in neuroblastoma (NB) tumorigenesis remains largely unexplored. High-risk NB tumors are often resistant to standard therapies, underscoring the need for novel treatment approaches. In many high-risk NBs, the MYCN oncogene is amplified, promoting an undifferentiated cell state associated with poor prognosis. Telomere maintenance is also predictive of survival in NB, with some tumors maintaining long telomeres through a telomeraseindependent process called alternative lengthening of telomeres (ALT), which is associated with disease persistence and frequent relapses. In this thesis, we optimized an m6A RNA-immunoprecipitation followed by sequencing (m6A-RIP-seq) method, allowing us to create the first m6A profile of NB tumors using low-input RNA samples. Applying m6A-RIP-seq in ALT-positive NB tumors, we uncovered an essential role for m6A-modified TERRA RNA in telomere maintenance. Our results suggest that m6A modification of TERRA RNA promotes its localization to telomeres in an hnRNPA2B1- dependent manner, forming condensate-like structures critical for TERRA’s function in ALT-positive NB. Furthermore, targeting the m6A methyltransferase METTL3, either alone or in combination with other therapeutic agents, led to significant telomere damage in ALT-positive NB cells. Additionally, we developed a novel MYCN-driven NB model by differentiating human embryonic stem cells into trunk neural crest cells (tNCCs) and then into sympathetic neurons. MYCN overexpression in this model recreated the undifferentiated state characteristic of NB. Using this model, we found that MYCN and m6A jointly regulate the progression from tNCCs to sympathoadrenal progenitor cells. MYCN overexpression disrupted the expression of m6A-related genes, contributing to an undifferentiated cell state as observed in NB. Analysis of the m6A profile in MYCN-driven NB tumors revealed that m6A regulates key NB-specific genes. Inhibiting METTL3 reversed the undifferentiated state induced by MYCN and synergized with chemotherapy to reduce tumor volume in MYCN patient-derived xenografts in vivo. In conclusion, our findings suggest that targeting the m6A epitranscriptome could provide a promising therapeutic strategy for high-risk NB, particularly in cases driven by ALT or MYCN amplification.