O-GlcNAcylation stabilized WTAP promotes GBM malignant progression in an N6-methyladenosine-dependent manner

Abstract Background: Interactions between mesenchymal glioblastoma stem cells (MES GSCs) and myeloid-derived macrophages (MDMs) shape the tumor-immunosuppressive microenvironment (TIME), promoting the progression of glioblastoma (GBM). N6-methyladenosine (m6A) plays important roles in the tumor progression. However, the mechanism of m6A in shaping the TIME of GBM remains elusive. Methods: Single-cell RNA sequencing and bulk RNA-seq datasets were employed to identify the critical role of WTAP in interactions between MES GBM and MDMs. The biological function of WTAP was confirmed both in vitro and in vivo. Mechanistically, mass spectrum, RNA immunoprecipitation (RIP), and co-immunoprecipitation assays were conducted. Results: Here, we identified that m6A methyltransferase Wilms' Tumor 1-Associated Protein (WTAP), whose protein stability could be synergistically enhanced via OGT-mediated O-GlcNAcylation and USP7-mediated de-ubiquitination, promoted LOXL2 m6A modification to enhance its mRNA stabilization in an IGF2BP2-dependent manner, upregulating secretion of LOXL2 protein (sLOXL2). sLOXL2 then interacted with integrin α5β1 on GSCs to activate FAK-ERK signaling, inducing mesenchymal transition of GSCs in an autocrine manner. Meanwhile, sLOXL2 also activated the integrin α5β1-FAK-ERK axis in MDMs, which promoted M2-like MDM phenotypes in a paracrine pathway, thereby contributing to T cell exhaustion to induce GBM immune escape. In translational medicine, combinations of the OGT inhibitor by targeting WTAP expression and the LOXL2 antagonist by disrupting MES GSC and MDM interactions showed favorable outcomes to the anti-PD1 immunotherapy. Conclusions: WTAP plays critical roles in mesenchymal transition of GSCs and formation of TIME, highlighting the therapeutic potential of targeting WTAP and its downstream effectors to enhance the efficacy of immunotherapy.