EXTH-45. Oncolytic Herpes Simplex Virus Synergizes with DNA-PKcs Inhibitors to Treat Glioblastoma

Glioblastoma (GBM) is an aggressive and lethal tumor with limited effective treatments. Recently, oncolytic herpes simplex virus (oHSV) G47Δ was approved in Japan for recurrent GBM. To enhance its therapeutic efficacy, we have explored combinations with inhibitors targeting DNA damage response (DDR). In a comprehensive screening of DDR inhibitors affecting non-homologous end joining (NHEJ), alternative NHEJ, homologous recombination (HR), and single-strand break repair pathways, we identified inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a critical regulator of NHEJ repair, as particularly promising. Inhibition of DNA-PKcs with M3814 (Peposertib), currently in clinical trials for GBM and solid tumors, synergized with two different oHSVs: G47Δ (γ34.5 and ICP47 deletions, ICP6 inactivation) and MG18L (Us3 deletion, ICP6 inactivation) in killing six patient-derived GBM stem-like cell (GSC) lines and four matched serum-cultured GBM cells, but was antagonistic in human astrocytes. This synergy was associated with degradation of DDR proteins, increased DNA damage and reduced repair, decreased stem-like cells, increased virus replication, cell cycle block, and enhanced cell death. To validate selectivity and restrict inhibition to GBM cells, we created GSCs with doxycycline-inducible DNA-PKcs shRNA, which significantly reduced sphere formation (Stemness). Intratumoral injection of oHSV into GSC tumors with transient doxycycline-induced DNA-PKcs knockdown significantly prolonged mouse survival compared to either monotherapy, achieving about 20-50% long-term survivors. Treatment of GSC-derived tumors with intratumoral oHSV and systemic M3814 also extended survival, highlighting the potential clinical utility of this strategy. These findings underscore the vulnerability of human GSCs to DNA-PKcs inhibition in the absence of DNA-damaging agents and its robust synergy with oHSV. Given that both G47Δ and M3814 have been evaluated in clinical trials, this study supports their continued clinical translation and further investigation into combination therapies for GBM.