Abstract
Amplification of epidermal growth factor receptor (EGFR) and active mutant EGFRvIII occurs frequently in glioblastoma (GBM) and contributes to chemo/radio‐resistance in various cancers, especially in GBM. Elucidating the underlying molecular mechanism of temozolomide (TMZ) resistance in GBM could benefit cancer patients. A genome‐wide screening under a clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 library is conducted to identify the genes that confer resistance to TMZ in EGFRvIII‐expressing GBM cells. Deep sgRNA sequencing reveals 191 candidate genes that are responsible for TMZ resistance in EGFRvIII‐expressing GBM cells. Notably, E2F6 is proven to drive a TMZ resistance, and E2F6 expression is controlled by the EGFRvIII/AKT/NF‐κB pathway. Furthermore, E2F6 is shown as a promising therapeutic target for TMZ resistance in orthotopic GBM cell line xenografts and GBM patient‐derived xenografts models. After integrating clinical data with paired primary–recurrent RNA sequencing data from 134 GBM patients who received TMZ treatment after surgery, it has been revealed that the E2F6 expression level is a predictive marker for TMZ response. Therefore, the inhibition of E2F6 is a promising strategy to conquer TMZ resistance in GBM.
Highlights
Glioblastoma (GBM) is the most aggressive primary brain tumor with high proliferation and invasion and easy recurrence after surgery.[1]
Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that these commonly differentially expressed sgRNAs were enriched in the spliceosome, ribosome, and cell cycle pathways (Figure 2a). We further analyzed these genes by ingenuity pathway analysis (IPA) and demonstrated their association with cancer, the cell cycle, and the DNA repair pathway in U87-EGFRvIII cells treated with TMZ for 14 days (Figure 2b; Table S2, Supporting Information)
We simulated the pathways revealed by IPA and found that the EGFRvIII/ PI3K/AKT/NF-κB and G protein-coupled receptor (GPCR)/ PLA/PKC/NFAT pathways are the most responsible for TMZ resistance in EGFRvIII-expressing GBM cells (Figure 2d; Table S4, Supporting Information)
Summary
Glioblastoma (GBM) is the most aggressive primary brain tumor with high proliferation and invasion and easy recurrence after surgery.[1]. Some new therapeutic strategies were emerging, such as systemic delivery of monoclonal antibodies,[4] temozolomide (TMZ) is the oral alkylating agent that serves as the current standard therapeutic for newly diagnosed GBM. TMZ reportedly causes cell cycle arrest in the G2/M phase and mediates DNA damage and, subsequently, apoptosis.[5] oral TMZ administration contributes to an overall increase in the survival of GBM patients, cancer cells eventually develop resistance to TMZ.[6] Currently, overcoming TMZ resistance remains a major challenge for GBM treatment. We focused on E2F6 because its expression was increased in EGFRvIII-overexpressing cells as determined by RNA-seq. Our results suggest that E2F6 inhibition is a promising therapeutic strategy for TMZ-resistant GBM
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