DYSREGULATION OF M6A RNA METHYLATION READERS AND ANTI-READERS, R-LOOPS AND THE DNA DAMAGE RESPONSE IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract AIMS Paediatric high-grade gliomas (pHGG) are one of the most devastating and incurable malignant brain tumours that have a very poor prognosis. The dysregulation of N6-methyladenosine (m6A) RNA methylation and R-loop homeostasis is associated with the development of cancers, including gliomas. We aimed to investigate whether m6A methylation reader and anti-reader proteins contribute to the pathogenesis of pHGG in order to identify possible therapeutic targets. METHODS To confirm whether m6A readers and anti-readers are upregulated at the mRNA and protein level, RT-qPCR and immunofluorescence was performed using patient-derived pHGG cell lines, control paediatric brain RNA and control human astrocytes. To investigate whether there is a link between m6A RNA methylation, R-loops and DNA damage, immunofluorescence was performed. siRNA knockdown experiments of m6A readers and anti- readers were performed to investigate effects on invasion, proliferation, apoptosis, global expression levels of m6A, R-loops and DNA damage response proteins, and neurosphere formation. RESULTS RT-qPCR and immunofluorescence revealed that m6A readers and anti-readers, such as fragile X mental retar- dation syndrome-related protein 1 (FXR1) and cell cycle associated protein 1 (CAPRIN1) are both significantly upregulated in pHGG cell lines at the gene and protein level. Immunofluorescence and colocalisation analy- sis revealed that m6A methylation, R-loops, γH2AX and ATM are significantly upregulated in pHGG cell lines, and that both FXR1 and CAPRIN1 are associated with R-loops to a high degree. Therefore, the upregulation of m6A effector proteins may contribute to the dysregulation of m6A methylation and R-loop homeostasis, and ulti- mately genomic instability. We also found that siRNA knockdowns of both FXR1 and CAPRIN1 reduce invasion, proliferation, and alter the global expression levels of m6A and R-loops. CONCLUSION Our findings suggest that FXR1 and CAPRIN1 are potential druggable targets for pHGG. In the future, therapeu- tics could be developed to target these two effector proteins and significantly improve the lives of patients with pHGG.