A non-canonical function for EZH2 in ARL13B-mediated therapeutic resistance

Glioblastoma stem cells (GSCs), a rare population of stem cells able to self-renew, are known to be responsible for GBM therapeutic resistance. Previously we have reported that chemotherapy initiates expansive cellular plasticity in GBM cells, which promotes the conversion of normal glioma cells to GSCs. Our initial investigation indicated that polycomb group protein EZH2 is critical for therapeutic stress-induced cellular plasticity. Chemical inhibition, as well as a shRNA-mediated knockdown, of EZH2 activity, prevents cellular plasticity-mediated conversions of non-GSCs to GSCs. To elucidate the molecular mechanisms underlying EZH2-mediated therapeutic resistance, gene expression analysis was performed in the presence of a specific EZH2 inhibitor. The results suggested that the most affected downstream target of EZH2 was ARL13B (13 fold change in expression relative to control), a member of the ADP-ribosylation factor-like family protein critical for cilia formation and maintenance. TCGA data analysis revealed that ARL13B expression is positively correlated with EZH2 expression (p<0.0001) and negatively correlated with survival of patients with grade III (p=0.03) and IV glioma (p=0.007). Additionally, Chip assay confirm enrichment of EZH2 on ARL13B promotor and knocking down of EZH2 by shRNA completely abolish the expression of ARL13B expression. These data indicated that therapy-induced EZH2 could regulate ARL13B expression post therapy in a non-canonical pathway. In order to investigate the role of ARL13B in vivo, shRNA-mediated knockdown ARL13B cells were intracranially injected to the mice. Mice bearing ARL13B knockdown tumors treated with TMZ showed significantly higher median survival compared to control shRNA-treated with TMZ. (p <0.0001). Immunofluorescent staining showed that ARL13B co-localize with primary cilium in primary GBM cells as well as in the patient-derived xenograft (PDX) model both in vitro and in vivo. The shRNA-mediated knockdown ARL13B cells fail to form primary cilium during anti-glioma chemotherapy, and therapeutic stress significantly enhanced formation (2 fold, p-value <0.0002) and length of primary cilium (1.5 fold, p<0.0001). Moreover, post-therapy recurrent PDX in the animal model showed 3-fold enhancement of cilia formation (p-value <0.0005). Moreover, therapeutic stress also induced sonic hedgehog (Shh) signaling in the PDX GBM. Shh activators molecules (ex. Smo, Gli) that are required for activation of Shh signaling were co-localized with the primary cilium. These observations suggest that therapeutic stress induced ARL13B expression and formation of the primary cilium, which can regulate Shh signaling during chemotherapy and may contribute to promoting therapeutic resistance and disease recurrence.