Introduction: Our previous work using in vivo CRISPR screens in CD8 WT and CD8 knockout animals identified Chek2 as the top gene encoding a kinase contributing to resistance to PD-1 blockade immunotherapy in gliomas. Genetic depletion of Chek2 or pharmacological treatment to abrogate CHK2 protein dimerization and kinase activity led to enhanced antigen presentation, cytosolic DNA sensing-STING pathway activation, and synergism with immune checkpoint blockade, supporting our hypothesis that mutant CHK2 proteins deficient in dimerization and/or kinase activity will have similar immune effects as pharmacological inhibition of CHK2. The prevalence of CHEK2 mutations in glioblastoma (GBM) tumors varies based on cohort, from 4-5% in TCGA Nature 2008 and Brain Tumor PDXs (Clin Cancer Res 2020) to 30% in a Taiwanese GBM patient population. Among all the CHEK2 mutations, I157T, R145W, and 1100delC are the most commonly occurring germline pathogenic mutations with disrupted protein function.
Methods: We employed a homology directed repair CRISPR knock-in to generate I157T, R145W, and 1100delC CHEK2 germline variants in control U87 cells. Next, we performed immunoprecipitation (IP) and phosphoproteomic analysis to study the mechanism by which mutant CHK2 proteins exert their effects. Furthermore, we performed CD8+ T-cell-mediated tumor cell killing assays by co-culturing the CHEK2 variant knock-ins with human CD8 T-cells to investigate whether glioma cells are rendered susceptible to CD8 T-cell killing.
Results: The three CHEK2 germline variants were successfully generated. Sanger sequencing determined knock-in efficiency and homozygous mutations were obtained from single cell colonies. IP and phosphoproteomic analysis identified interactors of CHK2 mutant proteins. Furthermore, CD8+ T-cell mediated tumor cell killing assays showed differential responses of T-cells to the three CHEK2 germline variants.
Conclusion : This project provides proof-of-concept for disrupting CHK2 function to induce immune responses in gliomas and as a potential immunotherapeutic avenue for meningiomas and other CNS malignancies where CHEK2 may be mutated/deleted.
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