EXTH-69. AN ENGINEERED microRNA CLUSTER FOR BROAD TARGETING OF EPIGENETIC COMPLEXES IN GBM

摘要

Epigenetic deregulation is a hallmark of glioblastoma (GBM), causing tumor cells to be refractory to differentiation, hyperproliferative and resistant to multiple genotoxic insults, including radiation and TMZ-induced DNA damage. Analysis of TCGA data revealed a highly significant inverse correlation between the expression of a group of microRNAs belonging to the neural lineage, downregulated in GBM, and several target proteins with epigenetic functions, including EZH2, BMI1 and LSD1, known oncogenes upregulated in the tumor. Consequently, we engineered an artificial DNA sequence encoding 5 of the above microRNAs arranged as a genetic cluster, and delivered to human GBM stem cells by lentiviral infection. The artificial transgene correctly produced all 5 mature microRNAs, and resulted in profound downregulation not only of putative targets, but also of other non-target oncogenes with epigenetic functions like DNMT1 and MYC, suggesting a synergistic effect caused by the combination of microRNAs. This combination determined a much more marked phenotypic effect than each single microRNA, both in vitro, measured as cell proliferation, induction of differentiation and clonogenic capacity, and in vivo, resulting in more than double survival time in mice with intracranial GBM xenografts. After temozolomide treatment, 90% of GBM cells overexpressing the multi-microRNA cluster underwent apoptotic cell death, as compared to 10–15% observed with single microRNA upregulation. Importantly, this effect was independent of MGMT expression status. Finally, co-culture experiments showed that all 5 overexpressed microRNAs were actively transferred from transduced cells to non-transduced ones, retaining their biological function in the acceptor cells. This suggests the potential for a vector-mediated delivery of clustered microRNAs in the clinical setting. In conclusion, we show a new microRNA-based gene therapy approach for GBM, which takes advantage of small size, ease of genetic manipulation, broad molecular targeting and cell-to-cell diffusivity proper of microRNAs.