2565 - Radiation-Induced <em>HMGB2 </em>Cytoplasmic Translocation Associates with Enhanced Immune Response Pathways in Glioma

Purpose/Objective(s): High HMGB2 nuclear expression in glioma cells is linked to increased mitosis count and tumor progression. Although sh-RNA mediated HMGB2 knockdown (KD) reduced cell proliferation and radio-sensitized glioma cell lines, in clinical samples high HMGB2 expression assessed by immunohistochemistry identified patients with better response to RT+TMZ. HMGB2 is a paralog of HMGB1, a known damage-associated molecular pattern (DAMP), however its expression is more specific to glioma cells compared to normal brain cells. We hypothesized that the divergence between in vitro and clinical findings may be due to involvement of HMGB2 in immunogenic cell death (ICD) and aimed to evaluate HMGB2 subcellular localization and associated transcriptomic changes in glioma stem cells (GSCs) and differentiated cells (DCs) following radiation treatment.

Materials/Methods: SF188 cells with HMGB2 KD and respective controls (NC) were de-differentiated using neurobasal medium, totaling four groups (DC-NC, DC-KD, GSC-NC, GSC-KD), irradiated with 3Gy, collected after 1h and 4h, and compared with the non-irradiated cells. RNA was purified and analyzed with microarray. Principal component analysis (PCA) was performed to visualize similarities related to differentiation status (DCs vs GSCs) and HMGB2 expression (NC vs KD). Functional enrichment analysis was performed using genes with log2 transformed fold change >1 (FC=2) for Gene Ontology (GO) terms. Efficacy of the HMGB2 KD, expression of GSCs markers, and nucleus-cytoplasm differential expression were confirmed by immunoblot.

Results: 1) At baseline, DCs showed enrichment of cell adhesion and migration pathways; after radiation, positive regulation of immune system pathways were enriched in all DCs groups compared to their GSCs counterparts. 2) Before radiation, HMGB2 expression was predominantly nuclear (80% in DCs, 72% in GSCs). After radiation, HMGB2 cytoplasmic expression increased to 56% in DCs, associated with positive regulation of response to external stimulus/hypoxia pathways, and increased to 80% in the GSCs, associated with enrichment of pathways related to cytoplasmic and extracellular (EC) components (e.g.,“cytoplasmic vesicle”,“EC matrix”). HMGB2 cytoplasmic translocation was validated in other glioma cells. 3) In DC-KD cells, with lower HMGB2 expression compared to DC-NC cells, pathways related to negative regulation of immunity and cell adhesion were enhanced.

Conclusion: In glioma, HMGB2 cytoplasmic translocation enhanced immune response pathways after radiation, which were significantly downregulated in HMGB2 KD cells. This may partially explain the better response to treatment in patients with high HGMB2 expression in clinical studies. Our findings suggest that, while nuclear HMGB2 expression is associated with tumor cell proliferation, radiation-induced HMGB2 cytoplasmic translocation may be involved in ICD in gliomas. Further studies on the crosstalk between glioma cells and immune cells are underway.