Loss of CD99L2 Contributed to Temozolomide Resistance and Glioblastoma Tumorigenesis Based on Genome-scale CRISPR/Cas9 Screening

Introduction: Glioblastoma Multiforme (GBM) is a highly aggressive and fatal brain malignancy, with Temozolomide (TMZ) serving as the first-line chemotherapeutic treatment. However, over 50% of patients do not respond to TMZ, and the underlying mechanisms remain unclear. This study utilized the GeCKO library to identify novel genes involved in TMZ resistance and to explore their functions. Methods: Loss-of-function genes related to TMZ resistance in GBM cells were identified using the GeCKO library and Next-Generation Sequencing (NGS), validated by qPCR and CCK-8 assays. CD99L2 function was assessed through proliferation, migration, and EdU assays in U251 and U87 cells. Tumor samples from 55 stage IV GBM patients were analyzed to explore the correlation between CD99L2 expression and Progression- Free Survival (PFS). Results: GeCKO library screening identified seven genes associated with TMZ resistance. After validation, CD99L2 was confirmed as a key contributor to TMZ resistance. Knockdown of CD99L2 increased the IC50 of U251 and U87 cells by 1.39- and 1.54-fold, respectively. Conversely, CD99L2 overexpression reduced the IC50 by 0.52- and 0.58-fold. CD99L2 knockdown also promoted tumor proliferation and aggressiveness. Additionally, higher CD99L2 expression was associated with longer PFS in GBM patients (median PFS: 7.87 months vs. 2.7 months, P=0.0003). Discussion: The functions of CD99L2 remain poorly understood. A few studies have reported that CD99L2 may serve as an adhesion molecule modulating inflammatory responses. One study has shown that CD99L2 is highly expressed in the brain and affects neuronal excitability. These findings suggest that CD99L2 may play a positive role in the body's defense against glioma. Conclusion: This study demonstrated that CD99L2 knockdown promotes TMZ resistance and tumorigenesis in GBM, suggesting its potential as a novel biomarker for TMZ resistance. conclusion: This study found that knocking down CD99L2 could contribute to TMZ-resistance and tumorigenesis in GBM.