IMMU-21. Antiviral Therapy Reverses CMV-Induced Oncogenic Signaling and Enhances NK Cell Cytotoxicity in Glioblastoma with Associated PD-L1 Upregulation and Immune Modulation

Abstract BACKGROUND Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) pathogenesis by promoting stemness, angiogenesis and immune evasion. Clinical trials targeting CMV in GBM have shown preliminary promise, however, the molecular mechanisms remain unclear. We hypothesized that CMV enhances oncogenic signaling and immune resistance in GBM, and that antiviral therapy may reverse these effects and increase susceptibility to immune-mediated cytotoxicity MATERIALS AND METHODS Human GBM cell lines (U251, U87, U138, T98G, LN229) and normal astrocytes were infected with a mCherry-human CMV TB40 strain (MOI 0.5). Phenotypic assays including spheroid & colony formation were performed. A 60-plex cytokine panel and Western blotting were used to evaluate cytokine profiles & signaling pathway activation (IL-6/STAT3, Akt, SOX2, Survivin, p-RB). Co-culture experiments with NK-92 MI cells were conducted using CMFDA and EthD dyes to assess cytotoxicity. RESULTS CMV infection upregulated IL-6/STAT3, SOX2, Survivin, p-AKT, and p-RB, promoting GBM cell proliferation, stemness, and apoptosis resistance. Infected cells exhibited a tumor-supportive cytokine profile and, after ganciclovir treatment, reduced VEGF and Angiopoietin-2 levels. CMV-infected astrocytes also showed increased oncogenic signaling and colony formation, suggesting a role in early transformation. Ganciclovir partially reversed these effects, shifting cytokine expression toward an immune-permissive state. While NK cells alone failed to lyse GBM cells, ganciclovir-treated, CMV-infected cells showed significantly enhanced NK-mediated cytotoxicity. PD-L1 expression increased after CMV infection and remained elevated post-treatment, indicating immune modulation and potential for checkpoint blockade. CONCLUSIONS CMV infection reprograms both glioblastoma cells and normal astrocytes toward a more aggressive, immune-evasive phenotype through activation of IL-6/STAT3, p-AKT, SOX2, and angiogenic pathways. Ganciclovir treatment partially reverses these effects, reducing VEGF and Angiopoietin-2 levels, shifting cytokine profiles, and—critically—enhancing NK cell-mediated cytotoxicity. Persistent PD-L1 upregulation following infection suggests a novel immune checkpoint vulnerability, opening the door for synergistic antiviral and immunotherapy approaches. These findings identify CMV not only as a driver of tumor progression but as a tractable therapeutic target in GBM.