RDNA-01. Microrna DEGRADATION MEDIATED GENETIC HETEROGENEITY AS A NOVEL MECHANISM FOR TEMOZOLOMIDE RESISTANCE IN GLIOBLASTOMA

Temozolomide (TMZ), the standard-of-care chemotherapy for glioblastoma, is a DNA alkylating drug. At cytotoxic doses, TMZ Induces high levels of alkylated DNA, including O6 methyl-guanine (O6MG), ultimately triggering cell death. In sub-lethal levels, O6MGs undergo non-Watson-Crick pairing, facilitating mutagenesis and promote acquired resistance. Whether the cytotoxic or mutagenic effects of TMZ predominate is largely determined by the DNA repair enzyme, Methyl-guanine-methyl transferase (MGMT). MGMT restores TMZ-alkylated DNA into its native form. To date, studies of MGMT have largely focused on its expression in large populations of cells. Variation of MGMT expression at a single cell level and pertinence of this variation to acquired TMZ resistance remains poorly understood. In single cells derived from the same patient-derived glioblastoma line, we found that the cell-to-cell variability in MGMT mRNA expression differed by as much as 10-fold. The distribution of single cell MGMT expression approximated that of a normal distribution. Upon treatment with TMZ, the mean expression of MGMT increased by two-fold. Additionally, TMZ treatment increased the standard deviation of the MGMT expression distribution by ten-fold, causing significant “widening” of the range the MGMT expression. Both of these effects were caused by degradation of miR-181d, an MGMT suppressing miRNA. TMZ treatment triggered the activation of ATM- and Rad3- related (ATR) kinase, which in turn activated Polyribonucleotide nucleotidyltransferase 1 (PNPT1), an RNAse that ultimately degradation of miR-181d. Our results suggest that RNA degradation enhanced stochastic variation in MGMT expression, allowing a wider range of MGMT expression from which the optimally fit clone can emerge. This novel form of TMZ resistance can be suppressed by over-expression of miR-181d, suggesting the potential of microRNA delivery as a therapeutic strategy. Accordingly, miR-181d delivery combined with TMZ treatment effectively cured intracranial murine models of patient derived glioblastoma xenografts (PDX) that expressed high levels of MGMT.