Glioblastoma (GBM) is one of the most lethal forms of brain cancer, and is known to be an aggressive, immunologically cold tumor, resulting in a lack of T-cell infiltration and killing. Thus increasing immune-mediated killing of GBM is a promising therapeutic method. Our group has found that Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase that has been shown to enhance tumor immunogenicity in glioblastoma, does so by inducing immunogenic cell death. We used LB-100, a small molecule inhibitor of the catalytic subunit of PP2A, as a promising drug-therapy. CRISPR-KO of PP2A was generated in murine glioma lines GL261 and SB28. Markers of immunologic cell death, such as calreticulin, gamma-H2AX, and HMGB1 were assessed. In vitro, western blotting of SB28 and GL261 showed significantly increased expression of gamma-H2AX in PP2A-KO compared to WT. Flow-cytometry staining of SB28 and GL261 revealed a dramatic increase in calreticulin expression in PP2A-KO compared to WT. An antibody-based fluorescence assay for HMGB1 also showed dramatic increase in PP2A-KO compared to WT. All experiments were repeated by treating LB-100 for 24 hour to SB28 and GL261 WT and PP2A-KO cells, and revealed a dose-dependent increase in expression of immunogenic cell death markers. In vivo, B6 mice were injected intracranially with SB28 WT or PP2A-deficient tumor and monitored till endpoint. At the endpoint, tumors were harvested, cryosectioned, and stained for calreticulin and gamma-H2AX. Mice injected with PP2A-KO tumor survived longer than WT, and had the highest expression of both calreticulin gamma-H2AX when compared to WT tissue. In conclusion, PP2A-deficiency and inhibition via LB-100 is demonstrated to induce immunogenic cell death in tumors, thus being a promising target for immunotherapy.
