The epitranscriptomic analysis of glioblastoma with and without drug resistance

Glioblastoma (GBM) is the most common, aggressive, and deadly type of malignant brain tumor. Despite the standard of care treatment including maximal surgical resection of the tumor, radiation, and chemotherapy, the median survival rate of GBM patients is only 15 months with a 5-year postdiagnosis survival of 5.8 %. Resistance to chemo or radiotherapy develops with GBM reoccurrence. While much has been uncovered regarding GBM biology in recent years from the mass of multi-omics studies, they have unfortunately failed to translate to clinical success. A new field of study, epitranscriptomics, was created in part by the discovery that RNA modifications have reader, writer, and eraser enzymes indicative of regulatory potentials. These evolutionarily controlled modifications maintain cellular functions and thus overall health. The dysregulation of specific RNA modifications and their associated enzymes in disease states, suggests their importance. However, there are no reports to date regarding the association of specific profiles of RNA modifications in cancer or GBM. In this work, a novel and accurate LC-MS/MS standard-free quantitation of ribonucleosides that allows for 81% coverage of the epitranscriptome of glioblastoma with 96% accuracy is detailed and validated. The modulation of the GBM epitranscriptome to one of temozolomide (TMZ) resistance is delineated in vitro and in vivo. The upregulation of N6-methyl-N6-threonylcarbamoyladenosine (m6t6A) is identified as a target biomarker in GBM resistant to TMZ. Knockdown of the specific writer gene for the RNA modification m6t6A, tRNA methyltransferase O (TRMO), shows promise for resensitization to TMZ treatment in preclinical GBM models. Following RNA isolation, purification, and fractionation, the upregulation of m6t6A is shown to be localized to the tRNA of GBM model LNZ308 with induced resistance to TMZ