Targeting CHEK2-YBX1&YBX3 regulatory hub to potentiate immune checkpoint blockade response in gliomas

Although the immunosuppressive microenvironment of glioblastoma (GBM) is well established, the mechanisms underlying the tumor’s resistance to CD8⁺ T cell-mediated cytotoxicity remain incompletely understood. Our previous work identified Checkpoint Kinase 2 (Chek2) as a key mediator of CD8⁺ T cell resistance in murine glioma using an in vivo CRISPR screen. Furthermore, we demonstrated that pharmacological inhibition of Chek2, when combined with PD-1/PD-L1 immune checkpoint blockade, markedly enhanced CD8⁺ T cell-mediated tumor killing and improved survival outcomes in preclinical models.

In the present study, we sought to investigate the immunosuppressive role of Chek2. Immunoprecipitation followed by mass spectrometry and phosphoproteomic analysis revealed that Chek2 interacts with the DNA/RNA-binding proteins YBX1 and YBX3, both known to repress transcription of pro-inflammatory genes. Single-gene knockout and overexpression studies of CHEK2, YBX1, and YBX3 across multiple glioma cell lines showed that these proteins mutually upregulate one another, forming a regulatory hub.

Integrated RNA sequencing and chromatin immunoprecipitation sequencing (ChIP-seq) identified a shared set of inflammatory genes suppressed by the CHK2-YBX1&YBX3 complex. Targeting YBX1 with the small-molecule inhibitor SU056 led to destabilization and degradation of the CHK2-YBX1&YBX3 hub, resulting in increased antigen presentation and enhanced antigen-specific CD8⁺ T cell proliferation. Importantly, the combination of SU056 with immune checkpoint blockade therapy significantly extended survival in multiple glioma models.

Taken together, these findings uncover a novel immunosuppressive mechanism driven by the CHK2-YBX1&YBX3 axis. Therapeutic disruption of this regulatory hub, particularly in conjunction with immune checkpoint blockade, holds promise for improving immunotherapeutic outcomes in glioma.