Abstract 948: PAX3-FOXO1-driven oncolytic herpes virus designed for treatment of alveolar rhabdomyosarcoma

Abstract Background: Alveolar rhabdomyosarcomas (aRMS) often express an oncogenic fusion protein PAX3-FOXO1. PAX3-FOXO1 (P3F) acts as an oncogenic transcription factor, driving aggressive and lethal tumors in children and young adults. P3F has proven elusive to small molecule inhibitors, and the low immunogenicity of aRMS tumors has impaired efforts to treat with immune checkpoint blockade. The absence of an effective targeted treatment for fusion-positive aRMS represents an important an unmet therapeutic need. Methods: We designed a system to combine and compare sequences which bind PAX3-FOXO1 to drive a transgene reporter system. We then used this system in a variety of rhabdomyosarcoma cell lines which either expressed PAX3-FOXO1, or were fusion negative. We then inserted this P3F-driven promoter into the oncolytic herpes simplex virus (oHSV) G47∆, driving the cytotoxic ICP34.5 gene, and tested its ability to kill P3F-positive and P3F-negative aRMS cell lines in vitro. To determine the effects of P3F expression on tumor cell response to oHSV infection, we performed RNA sequencing and differential gene expression using DESeq2 to assay alterations between P3F-positive and negative tumor cells during the lifetime of oHSV infection. Enrichment analysis on genes identified as significantly different were then analyzed via EnrichR to evaluate gene ontology and pathways significantly enriched during infection which differed based on PAX3-FOXO1 expression. Results: We identified two synthetic promoters which exhibited expression of luciferase or GFP reporter genes only in the presence of P3F (T-Test p<0.05, n=10). We then inserted the cytotoxic HSV1-ICP34.5 gene into the ICP6 gene of the previously validated oHSV G47∆ under this synthetic promoter. These novel G47∆-P3Fpro:ICP34.5 elicited quicker cell killing in several P3F-positive RMS lines faster than the parental virus, while exhibiting the same cell killing kinetics in matched fusion negative cells (2-way ANOVA p<0.05, n=3, Tukey multiple comparisons test). Differential gene expression over time demonstrated PAX3-FOXO1 is involved in modulating a substantial amount of immune response genes during oHSV infection including IFI16, SAMD9, and DUSP4. Expression was also significantly altered between fusion positive cells infected with the ICP34.5-armed virus compared to fusion negative cells infected with the same virus. Conclusions: We demonstrate the synthesis of a novel PAX3-FOXO1-controlled gene expression system, and its implementation within an oHSV to combat PAX3-FOXO1 fusion oncogene-positive aRMS. Our study demonstrates the influence of PAX3-FOXO1 during viral infection with an oHSV and the potential to tailor P3F specific promoters to novel cancer targeting vectors. Citation Format: Cole W. Peters, Theodore Nowicki, Moe Kawakami, Connor Kidd, Analynn Lechein. PAX3-FOXO1-driven oncolytic herpes virus designed for treatment of alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 948.