Epitranscriptome-wide Approach for Identifying Specific Writer of RNA Modification as Knockdown Target for Resensitizing Glioblastoma Cells to Temozolomide Treatment

Background: The resistance to temozolomide (TMZ) treatment remains a challenge in caring for many patients with glioblastoma (GBM) tumors. There is growing interest in unraveling the roles of epitranscriptome in cancer. Among all the known RNA modifications, 6 different modifications have been individually linked to GBM. In one of those earlier studies, Zhao and his associates reported TMZ treatment would induce higher expression of METTL3, which is one of the writers for N6-methyladenosine (m6A) modification. By combining the TMZ treatment with METTL3 silencing, the growth of TMZ-resistant xenograft was suppressed. To achieve untargeted epitranscriptome-wide analysis, our group has recently developed a mass spectrometric method that can accurately quantify each RNA modification despite its identity. Aim: In this pilot study, we aim to use an epitranscriptome-wide approach to identify a new potential target for alleviating the resistance of GBM cells to TMZ treatment. Methods and Results: In this study, patient-derived GBM cell lines that were classified as TMZ-sensitive or TMZ-resistant were chosen as in vitro models. Using our developed method, the epitranscriptomes of selected GBM cell lines were profiled. By comparing the epitranscriptomic profiles, upregulated RNA modifications were identified and correlated to the TMZ resistance. One of the most upregulated modifications is N6-methyl-N6-threonylcarbamoyladenosine (m6t6A). The association of m6t6A upregulation to TMZ resistance was confirmed by comparing the epitranscriptomes in xenografts with either primary or recurrent GBM cells. Aiming to resensitize TMZ-resistant GBM cells, the writer for m6t6A modification, known as tRNA methyltransferase O (TRMO), in selected TMZ-resistant GBM cell cultures was knocked down. The resulting cells were resensitized to lower dosages of TMZ. Conclusion: This pilot study demonstrates the use of comprehensive epitranscriptomic profiling is a feasible approach for identifying specific RNA writer of RNA modifications as a knockdown target for resensitizing GBM cells to TMZ treatment.