M6A RNA METHYLATION AS A THERAPEUTIC TARGET IN HIGH GRADE GLIOMA

Abstract

Abstract AIMS High grade glioma (HGG) is a devastating brain cancer that has poor prognostic outcomes. Very recently, the field of epitranscriptome has emerged as a novel druggable target for multiple diseases, including cancers. In the present study, we sought to investigate the functional importance of N6-methyladenosine (m6A) RNA methylation in the pathogenesis and drug resistance of paediatric and adult high grade glioma. METHOD To identify the expression of key m6A effectors, RT-qPCR and immunofluorescence were undertaken in patient derived HGG cell lines, primary low passage HGG cells and human tissues. The expression of these effectors was modified using siRNA-mediated knockdown and small molecules to identify their effects on key transcription factors. The consequences of modification on the ability of the cells to form neurospheres and invade was also assessed. Developing chemotherapy-resistant cell lines is undertaking to assess the effect of m6A RNA levels on the ability of HGG cells to adapt to environmental changes. Measuring the level of m6A-modified transcripts were achieved by developing LC-MS/MS and MeRIP (methylated RNA immunoprecipitation) qPCR techniques. RESULTS We found that key m6A effectors such as methyltransferase like 3 (METTL3), Wilms tumor 1-associating protein (WTAP) and fat mass and obesity-associated (FTO) are expressed at significantly higher level in paediatric and adult HGG models than in control at both gene and protein levels. Furthermore, these effectors regulate the expression of key transcription factors in self-renewal, cell cycle regulation and tumorigenesis. siRNA-mediated depletion of m6A effectors and inhibition of METTL3 using small molecules such as STM2457 and STM3006 reduce the capacity of cells to form neurospheres and invade as well as improving the sensitivity of HGG cells to chemotherapeutic agents. CONCLUSION Our findings suggest that m6A effectors are potential pro-oncogenic factors that, if druggable, will represent an advanced avenue in HGG therapy.