Background: Glioblastoma (GBM) exhibits profound resistance to CD8⁺ T cell-mediated killing, yet the tumor-intrinsic mechanisms driving this immune evasion remain poorly defined. Our earlier study revealed Checkpoint Kinase 2 (Chek2) as the driver of CD8+ T cell resistance. This study investigates the immunomodulatory program exerted by the CHK2-YBX1&YBX3 regulatory hub.

Methods: Protein-protein interactions were investigated through immunoprecipitation (IP) followed by mass spectrometry (MS) and phosphoproteomics. Single gene knockout of CHEK2, Y-box-binding protein 1 (YBX1), and Y-box-binding protein 3 (YBX3) were generated in human and mouse glioma cells. Transcriptomic and epigenetic alterations were characterized by bulk RNA sequencing and chromatin immunoprecipitation sequencing (ChIP-seq). Single-cell RNA sequencing and spatial transcriptomics analysis were performed to evaluate CHK2-YBX1&YBX3 related phenotype in human GBM tumors. In vivo survival studies were conducted to assess the therapeutic potential of CHK2-YBX1&YBX3 degradation and immune checkpoint blockade (ICB).

Results: CHK2, YBX1, and YBX3 exhibited reciprocal positive regulation and depletion of any of these genes resulted in derepression of pro-inflammatory gene expression. Pharmacological inhibition with the drug targeting YBX1 led to degradation of the CHK2-YBX1&YBX3 hub accompanied by enhanced antigen presentation and antigen-specific CD8⁺ T cell proliferation. Combination therapy targeting CHK2-YBX1&YBX3 hub and ICB significantly improved survival in preclinical glioma models.

Conclusions: These findings define a novel glioma-intrinsic immunosuppressive program and proposes targeting the CHK2-YBX1&YBX3 hub to potentiate response to ICB in glioma.