m6A RNA Methylation Is Increased in Tumour Invasive Regions and Influences Invasive Capability and Chemotherapeutic Sensitivity in Adult Glioblastoma

Adult glioblastoma multiforme (GBM) is the most common primary malignant brain tumour caused by multiple molecular factors. N6-methyl-adenosine (m6A) is an abundant RNA modification that governs cellular RNA metabolism. We hypothesise that changes in m6A-modified RNA and regulatory machinery such as the writer proteins, Methyltransferase 3 (METTL3) and WT1-associating protein (WTAP), the demethyltransferase protein, and Alpha-ketoglutarate dependent dioxygenase (FTO), are driving factors of GBM development and treatment resistance. Here, we investigated m6A-RNA spatial and quantitative abundance and expression of m6A effector proteins directly in GBM tissue and patient-derived low-passage primary adult GBM and low-grade glioma (LGG) cells, and explored the consequences of m6A-RNA disruption on GBM invasive capabilities, self-renewal and responsiveness to temozolomide (TMZ). We observed that METTL3, WTAP and FTO transcript and protein expression were significantly increased in cells derived from invasive regions of GBM tumours, and elevated WTAP and FTO expression significantly correlated with poor GBM patient survival. We further found that the abundance of m6A-modified RNA in GBM tumours was significant higher in rim and invasive tissue, as well as significantly higher in patient-derived cells from GBM tumour invasive regions. Functional depletion of these effector proteins significantly altered m6A levels on and the expression of the pluripotency stem cell marker SOX2 while also impairing self-renewal and cell invasion behaviour and increasing sensitivity to TMZ. The targeting of RNA modification regulatory mechanisms reveals novel therapeutic strategies aimed at improving clinical outcomes for GBM patients.