Targeting The BHLH Transcriptional Factor Olig2 In Murine Model Of Glioblastoma To Study The Effect On Tumor Initiation,Progression And Radiosensitivity

Objective Glioblastoma is the most common malignant brain tumor in adults and is notoriously insensitive to radiation and genotoxic drugs. Four core TFs-POU3F2, SOX2, SALL2 and OLIG2-whose induction is sufficient to reprogram differentiated glioblastoma cells into stem-like cells capable of in vivo tumor propagation. Among them, the lineage restricted bHLH transcription factor Olig2 is expressed in replication competent neural progenitors and also in the tumor initiating cells of human glioblastomas. The aim of this study was to test whether genetic suppression of Olig2 could slow down glioblastoma progression and increase radiosensitivity in vivo.Methods Tumor initiating cells from freshly isolated human gliomas were transduced with lentiCRISPR vector to knockout Olig2 and stereotactically implanted into the brains of nude mice. P53 mutant gliomas provided an internal negative control. Then we exploited inducible CRISPR to achieve conditional knockdown of Olig2 by doxycycline. And then BT145 and BT74 transduced with the inducible CRISPR construct were intracrinial injected to the SCID mice to explore the effect of Olig2 knockdown on tumor progression in vivo. In order to test whether genetic suppression of Olig2 could sensitize p53-positive human gliomas to radiotherapy in vivo, radiation-induced expression of p21 and radiosensitivity were firstly tested in human glioblastoma cells with and without Olig2 in vitro. Once human glioblastoma murine model was established, the mice were divided into 4 groups:control group, Olig2 knockdown group, radiation group (10Gy,5 fractions), and Olig2 knockdown plus radiation group. The mice were imaged biweekly for luciferase activity to monitor tumor progression.Results Olig2 was essential for intracranial tumor formation with p53 wild type BT145 murine model of glioma, but not required for the p53 mutant BT74 murine model. Inducible CRISPR could knock down Olig2 efficiently. And Olig2 knockdown could slow down brain tumor progression both in p53 wild type BT145 murine model and in p53 mutant BT74 murine model. Olig2 opposed radiation-induced expression of p21 and promoted radioresistance in p53-positive human glioma cells (34.5%±2.4% cell growth decrease in Olig2 knockout cells vs 15.6%±1.3% in Olig2 wild type cells, p=0.002). BT145 cells expressing the inducible Olig2 CRISPR were then injected into mice brains. And the mice received both Olig2 knockdown and 2Gy*5 radiation treatments showed increased survival time than mice that received radiation alone or Olig2 knockdown alone, as shown by Kaplan-Meier curve. Furthermore, BT145 cells transduced with inducible Olig2 CRISPR and treated with 2Gy*5 radiation showed increased levels of cleaved caspase 3 compared with radiation or Olig2 knockdown alone by Western blot.Conclusion Olig2 is essential for intracranial tumor formation and genetic suppression of Olig2 can sensitize p53-positive human gliomas to radiotherapy in vivo. Towards the long-term goal of exploiting Olig2 as a therapeutic target for glioma, a "proof of concept" demonstration of in vivo radiosensitization will be an essential component of our pre clinical database.