Oncolytic herpes simplex virus, M002, increases the antitumor radiation response by decreasing the G2/M arrest in murine rhabdomyosarcoma orthotopic allograft cells

Introduction: Rhabdomyosarcoma (RMS) represents the predominant soft tissue sarcoma in children. For most RMS patients, multimodality therapy is employed to achieve optimal outcomes, incorporating chemotherapy, radiotherapy (RT) and surgery. RT, although an integral component of local control, often results in significant long-term sequelae. Further, children with RMS who relapse after RT continue to experience the poorest outcomes. These issues underscore the need to develop strategies to minimize late toxicities and improve therapeutic index. Investigations have indicated that RT-induced antigen release amplifies the immune response triggered by oncolytic viruses (OVs), and viral infection of tumor cells impedes the repair of RT-induced DNA damage, both enhancing RT efficacy. We previously showed that treatment of murine orthotopic allograft (OA)-RMS cells with an oncolytic Herpes Simplex Virus (oHSV), M002, enhanced the response to RT. These findings led us to investigate the mechanism by which M002 enhances the antitumor response to RT in OA-RMS cells. Methods: Murine OA-RMS cells were treated with M002 (0 or 5 plaque forming units (PFU)/cell) for 24 hours and whole cell RNA sequencing was performed. RT doses were delivered as X-rays at 275 kV and 10.0 mA. Viability was measured using almarBlue. Cell cycle distribution was assessed by flow cytometry. Summary of results: OA-RMS cells demonstrated resistance to RT as measured by no significant change in viability between control cells and RT treated cells on post-treatment day 6. RNA sequence data and pathway analysis of M002 (5 PFU/cell) treated OA-RMS cells showed significant downregulation of genes involved in the G2/M pathway. Combined treatment with 2 Gy RT and M002 abrogated G2/M phase arrest caused by RT alone. The percentage of OA-RMS cells arrested at the G2/M phase increased after 2 Gy RT to 24.4% compared to 8.3% in the control cells. Combined treatment with M002 and RT decreased the percentage of OA-RMS cells arrested at G2/M phase to 2.5%. Combining M002 with RT significantly increased the sub-G1 population compared to the control group, indicating dying and apoptotic cells. Conclusion: We demonstrate that the oHSV, M002, significantly suppresses the genes regulating the G2/M phase of the cell cycle in OA-RMS cells. Combining RT with M002 reduced the G2/M phase arrest caused by RT. The loss of G2/M arrest was accompanied by an increase in cell death. These results indicate that M002 is a promising strategy to disrupt the G2/M checkpoint, a mechanism through which RT-resistant RMS tumors function.