TBID-armed oncolytic adenovirus KD01 engages a CDCA8-associated mitotic vulnerability for anti-glioma therapy

Glioblastoma is among the most lethal primary brain tumors, and the development of oncolytic viruses has been hampered by the lack of molecular cues that guide virus design and help identify tumors likely to respond. Methods: We engineered a tBID-armed oncolytic adenovirus derived from adenovirus type 5 (Ad5), designated KD01, by modifying the E3 region to express a humanized tBID fragment. KD01 activity was evaluated in human and murine glioma cell lines using CCK-8 assays, flow cytometry, and JC-1 staining with empty-backbone virus M20 as control. Global transcriptomic and proteomic analyses delineated signaling programs altered after infection. The functional relevance of CDCA8 was tested in glioma models with stable CDCA8 overexpression or knockdown. Antitumor efficacy was further examined in an immunocompetent CT2A orthotopic model, and intracranial safety was assessed in Syrian golden hamsters. Results: KD01 consistently induced stronger cytotoxicity than M20 and was associated with BID/tBID-BAX-BAK apoptotic signaling. Multi-omics analyses showed induction of pro-apoptotic programs together with suppression of CREB1 and the mitotic regulator CDCA8, affecting pathways involved in cell-cycle regulation, chromosome segregation, and oncogenic signaling. In glioblastoma datasets, CDCA8 was upregulated and inversely correlated with BID, although baseline CDCA8 expression did not independently stratify TCGA GBM survival. In vivo, CDCA8 knockdown enhanced, whereas overexpression blunted, KD01 efficacy. KD01 also reduced tumor burden and prolonged survival in CT2A-bearing mice and was well tolerated intracranially in hamsters. Conclusions: KD01 exerts potent anti-glioma activity with a favorable preliminary safety profile and is associated with suppression of a CREB1/CDCA8-linked proliferative program. These findings support CDCA8 as a functional determinant of KD01 responsiveness and an exploratory candidate response biomarker that requires validation in larger patient-derived model panels.