The Application Of The IBET System In Decoding The Pattern Of Potential Driver Mutations During The Intiation And Progression Of High-grade Glioma

Glioblastoma(GBM)is a devastating brain tumor,with a median overall survival(OS)of fifteen months.There is no effective treatment strategy that can significantly improve prognosis and the pathogenesis is unknown yet.A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of crucial importance.Here we developed an Intra Brain-Electropotation-Transposon-Based Conditional Gene Editing Mouse Model(iBET)to recapitulate human glioblastoma by introducing high frequency mutation combinations occurred in human GBMs into the conditional gene editing mouse model.Systematic validation shows that iBET model can specifically introduce mutations into neural stem cells(NSCs)and tdT reporter was expressed in those mutant cells based on Cre.And the number of initial mutant cell is small,with single-cell resolution,which facilitates the subsequent study of tumorigenesis progression by lineage tracing.From the sequencing data of 291 GBM patients in TCGA database,we identified 13 important mutation genes with the high frequency and more than 30 multiple mutation combinations with the high frequency.P53/NF1;P53/NF1/PTEN;CDKN2A/CDKN2B/EGFR/PTEN were among the first combinations selected to establish the corresponding iBET mouse models.Our results showed that all three mutation combinations could effectively drive tumorigenesis with iBET mouse model.P53/NF1 and P53/NF1/PTEN mutation combinations could effectively drive tumorigenesis in the iBET context,with 47%and 100%penetrence,116 days' and 230 days' median survival time,respectively;While the tumor formation efficiency of CDKN2A/CDKN2B/EGFR/PTEN combination is only 6%.When horizontally comparing the phenotypes between the P53/NF1 and P53/NF1/PTEN models,we found that the existence of the PTEN mutation greatly accelerated the process of tumorigenesis induced by P53/NF1 mutations.Our work also verifies the feasibility of utilizing the iBET system to study the function of tumor suppressors during tumor progression rapidly and efficiently.Compared with the conventional gene editing mouse model,the iBET mouse model greatly saves research resources.It possesses the advantages of short construction time and uniform background of laboratory animals.It makes it possible to horizontally compare different mutation combinations to study the functional maps of multiple genes in tumorigenesis in the same system.At the same time,on the basis of current research,we can use iBET model to map the function of GBM drivers in the natural microenvironment of mouse brain,and find out the relationship between the cell types and the drivers in the development of GBM by lineage tracing,so as to study the cell lineage and molecular transformation mechanism in the transitional stage of GBM.We foresee that the iBET will become a useful in vivo system to understanding tumor progression and developing new treatment methods.