Background: High grade glioma (HGG) is a devastating brain cancer with median survival of less than 18 months. The field of epitranscriptomics has emerged as a novel druggable target for multiple diseases, including cancers, with N6-methyladenosine (m6A) as the most prevalent mRNA modification. M6A levels are modified by ‘writers’ such as METTL3, METTL14 and WTAP and ‘erasers’ such as FTO and ALKBH5. The interplay between these two processes dynamically regulates the m6A modification. The fate of modified RNA is determined by the m6A-bound ‘readers’ such as YTHDF1 or YTHDF2 with rapid effects on translation and mRNA abundance. In the present study, we sought to investigate the functional importance of m6A RNA methylation in the pathogenesis and drug resistance of HGG.

Methods: RT-qPCR and immunofluorescence for m6A modulators were undertaken in patient-derived HGG cell lines, paediatric HGG cells and human HGG tissues. The expression of these modulators was modified using siRNA knockdown and small molecules (such as a novel METTL3 inhibitor) to identify effects on key transcription factors in HGG. The effect of modification on stemness behaviours such as the ability to form neurospheres was also assessed. Developing chemotherapy-resistant cell lines, 3d cell culture and hypoxic cell culture were undertaken to assess associations between m6A RNA levels and the ability of HGG cells to adapt to environmental change. Measuring the level of m6A-modified transcripts were achieved by developing a MeRIP (methylated RNA immunoprecipitation) qPCR technique. An LC-MS/MS based protocol was developed to quantify m6A levels on poly-A+-enriched RNA.

Results: We found that key m6A RNA methylation effectors such as METTL3, FTO and WTAP are expressed at significantly higher level in HGG cell lines and tissues than in control cells (astrocytes) at both gene and protein levels. LC-MS/MS and immunofluorescence showed an increase in the level of m6A in paediatric HGG compared with normal brain. Furthermore, knockdown of METTL3, FTO and WTAP affects the expression of key transcription factors in self-renewal, cell cycle regulation and tumorigenesis including FOXM1, nestin and SOX2. Depletion of m6A mediators also reduces the ability of cells to form neurospheres, increases apoptosis and suppresses invasion. Moreover, chemical inhibition of METTL3 and FTO was associated with a downregulation in the expression of oncogenes, reduction in neurosphere size and increase in apoptosis. Inhibitors of m6A effectors also sensitize HGG cell lines to the effect of chemotherapeutic agents such as temozolomide. LC-MS/MS data revealed dose-response change in m6A levels in cells treated with different concentrations of these inhibitors.

Conclusion: These findings suggest a key role for RNA methylation in phenotypic plasticity in HGG, and could represent a new avenue for developing effective treatment strategies, with targeted inhibition of METTL3 showing promise.

Citation Format: Masar Radhi, Jonathan Rowlinson, Nigel Halliday, Simon Deacon, Ella Collinson, Helen Knight, Dong-Hyun Kim, Stuart James Smith. m6A RNA methylation as a therapeutic target in high grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2234.