Purpose: Oncolytic herpes simplex virus-1 (oHSV) infection of brain tumors activates NOTCH, however the consequences of NOTCH on oHSV-induced immunotherapy is largely unknown. Here we evaluated the impact of NOTCH blockade on virus-induced immunotherapy. Experimental Design: RNA sequencing (RNA-seq), TCGA data analysis, flow cytometry, Luminex- and ELISA-based assays, brain tumor animal models, and serum analysis of patients with recurrent glioblastoma (GBM) treated with oHSV was used to evaluate the effect of NOTCH signaling on virus-induced immunotherapy. Results: TCGA data analysis of patients with grade IV glioma and oHSV treatment of experimental brain tumors in mice showed that NOTCH signaling significantly correlated with a higher myeloid cell infiltration. Immunofluorescence staining and RNA-seq uncovered a significant induction of Jag1 (NOTCH ligand) expression in infiltrating myeloid cells upon oHSV infection. Jag1-expressing macrophages further spread NOTCH activation in the tumor microenvironment (TME). NOTCH-activated macrophages increased the secretion of CCL2, which further amplified myeloid-derived suppressor cells. CCL2 and IL10 induction was also observed in serum of patients with recurrent GBM treated with oHSV (rQnestin34.5; NCT03152318). Pharmacologic blockade of NOTCH signaling rescued the oHSV-induced immunosuppressive TME and activated a CD8-dependent antitumor memory response, resulting in a therapeutic benefit. Conclusions: NOTCH-induced immunosuppressive myeloid cell recruitment limited antitumor immunity. Translationally, these findings support the use of NOTCH inhibition in conjunction with oHSV therapy.

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