P01.02.b Trna Modifications, the New Frontiers in Glioblastoma Therapy

BACKGROUND Efforts to develop glioblastoma (GB) therapies based on identified genomic or transcriptomic phenotypes have eluded scientists for decades. In recent years, the epitranscriptome, i.e. RNA modifications, have emerged as interesting targets for cancer therapy. tRNA modifications have garnered interest for their role in cancer biology. However, in GB, the role of tRNA modifications in regulating oncogenesis and mRNA translation remains incompletely explored. MATERIAL AND METHODS High throughput mass spectrometry (LC-MS/MS) was used to identify tRNA modifications in surgically collected glioma samples of varying grades. CRISPR/Cas9 mediated KO was used to study and characterize enzymatic systems responsible for modifications of interest. RNA-seq and Ribo-seq were used to decipher the impact of tRNA modifications on codon decoding and mRNA translation. Animal models of glioma were used to study glioma heterogeneity and the impact of tRNA modifications in vivo. scRNA-seq was further used to analyze animal models of transgenic glioma cells. RESULTS LC-MS/MS was able to reveal >20 significantly enriched tRNA modifications in high grade glioma (GB and GIV Astrocytoma) vs low grade glioma (GII & III Astrocytoma and oligodendroglioma). We focused on a modification system mediated by the tRNA dioxygenase ALKBH1 which mediates the oxidation of m5C to hm5C and further to f5C in cytosolic and mitochondrial tRNA. CRISPR/Cas-9 KO and plasmid mediated stable overexpression were used to study ALKBH1 function in 5 different glioma cell lines. RNA-seq and Ribo-seq revealed the impact of ALKBH1 on codon decoding and translation of neuronal linked pathways. ALKBH1 induced a stem-cell line phenotype in glioma cells, led to slower proliferation in vitro and slower protein synthesis. On the other hand, in vivo ALKBH1 led to higher proliferation, immune evasion, and more heterogeneity. scRNA-seq analysis revealed reduced immune cell infiltration and a neuronal phenotype induction in ALKBH1 overexpressing glioma cells. CONCLUSION tRNA modifications regulate various oncogenic processes in GB and are linked to aggressiveness and outcome. Targeting tRNA modifications could prove a viable and successful strategy for GB therapy.