Loss of CD99L2 contributed to temozolomide resistance and glioblastoma tumorigenesis based on Genome-scale CRISPR/Cas9 screening

Abstract

Abstract Introduction: Glioblastoma multiforme (GBM) is a highly aggressive and fatal malignancy of central nervous system. Temozolomide (TMZ) is the first-line chemotherapeutic drug for GBM, although drug resistance remains challenging. To date, MGMT promoter hypomethylation explains part of TMZ resistance cases. The purpose of this study was to utilize the whole human Genome-scale CRISPR-Cas9 knockout (GeCKO) library to screen for novel genes involved in TMZ resistance and explore their functions. Methods: A GeCKO library combined with next-generation sequencing (NGS) was used to screen loss-of-function genes conferring TMZ resistance in GBM cells, then candidate genes were filtrated by qPCR and CCK-8 analysis. Cell proliferation assay, colony formation, wound healing, transwell, and 5-ethynyl-2’-deoxyuridine (EdU) assays were performed to explore the roles of CD99L2 on glioblastoma tumorigenesis in U251 and U87 cell lines. Tumor samples from stage IV GBM patients were used to assess the correlation between CD99L2 expression and progression-free survival (PFS) of the patients. Results: Seven candidate genes including USP17L20, CD99L2, IL2RG, STEAP4, ALKBH3, EDARADD, and PPAPDC3 were identified. Among them, CD99L2 had been confirmed to contribute to TMZ resistance. In U251 and U87 cells treated with TMZ, the knockdown of CD99L2 improved IC50 by 1.39 and 1.54 times compared with the NC groups, respectively. The overexpression of CD99L2 reduced IC50 by 0.52 and 0.58 times compared with the vector groups. CD99L2 knockdown also enhanced tumor proliferation, aggression, vice versa. Moreover, the patients with high-expressed CD99L2 were associated with longer PFS (median PFS: 7.87 months vs. 2.7 months, P = 0.0003). Conclusion: This study found that knocking down CD99L2 could contribute to TMZ-resistance and tumorigenesis in GBM.