M6A RNA METHYLATION AS A THERAPEUTIC TARGET IN HIGH GRADE GLIOMA

Abstract AIMS High grade glioma (HGG) is a devastating brain cancer. The field of epitranscriptome has emerged as a novel druggable target for multiple diseases. N6-methyladenosine (m6A) is the most prevalent mRNA modification. It is co-transcriptionally installed by a methylase complex called “writer” composed of METTL3, METTL14 and WTAP. The modification is reversible and erased by FTO and ALKBH5 which are called “eraser”. The fate of the modified RNA is determined by the m6A-bound “reader. Here, we sought to investigate the functional impor- tance of m6A RNA methylation in the pathogenesis and drug resistance of HGG. METHODS To identify the expression of key modulators of RNA methylation, RT-qPCR and immunofluorescence were un- dertaken HGG cells and tissues. The expression of these modulators was modified using siRNA knockdown and small molecules to identify their effects on key transcription factors. The consequences of m6A RNA methy- lation disruption on the invasive capabilities, self-renewal and sensitivity to temozolomide was also assessed. Measuring the level of m6A-modified transcripts were achieved by developing MeRIP (methylated RNA immuno- precipitation) qPCR technique and LC-MS/MS on poly-A+-enriched RNA. RESULTS METTL3, WTAP and METTL14 are highly expressed in in HGG cells and tissues. LC-MS/MS and MeRIPqPCR showed an increase in the level of m6A in HGG. Furthermore, knockdown of m6A effectors affects the expression of key factors in self-renewal, cell cycle regulation and tumorigenesis including FOXM1, nestin and SOX2. It also reduces the ability of cells to form neurospheres, suppresses invasion and improves the sensitivity of HGG cells to temozolomide. Inhibiting the enzymatic activity of METTL3 using STM2457 and STM3006 reduces the ability of HGG cells to invade and form neutrospheres and also sensitizes the HGG cells to the effect of temozolomide. CONCLUSION These findings suggest the role of RNA methylation in the pathogenesis of HGG, and therefore, could open a new avenue for developing effective treatment strategies.