Oncolytic HSV-1-Mediated JAG1 Blockade Induces Glioma Senescence-Associated Secretory Phenotype to Increase Macrophage Activation and Cetuximab-Mediated Senolysis

Abstract Oncolytic HSV-1 (oHSV) treatment induces Notch signaling and myelosuppression in the tumor microenvironment (TME) of preclinical cancer models. Clinically, the Notch ligand JAG1 was upregulated in recurrent high-grade glioma patients treated with the oHSV CAN-3110 and correlated with poor prognosis. To better understand endogenous JAG1-mediated signaling in glioma cells and tumor-associated macrophages (TAMs), we engineered a JAG1-antagonizing oHSV (OD-0J1) and interrogated its impact on cancer and myeloid cells in the tumor microenvironment. OD-0J1 antagonized JAG1-mediated Notch signaling and suppressed tumor growth in athymic nude and humanized mice, an effect reliant on Notch signaling in tumor cells. Kinome profiling revealed that OD-0J1 treatment suppressed CDK1, resulting in activation of the G2/M cell cycle checkpoint. Cell cycle arrest led to senescence and correlated with increased reactive oxygen species, p62 and autophagosome accumulation, and senescence-associated β-galactosidase activity. OD-0J1-induced senescence resulted in increased production of inflammatory chemokines and DAMPs, such as IL-1β, HMGB1, and extracellular ATP. Co-culturing macrophages with OD-0J1-infected tumor cells led to stimulation of chemotactic and pro-inflammatory pathways, as well as increased Fc receptor activation. Single-cell RNA sequencing and flow cytometric analysis of F4/80+ cells isolated from tumors showed a shift from tumor-supporting TAMs to inflammatory macrophages upon OD-0J1 treatment. Heightened EGFR activation in senescent cells was a mechanism to escape cell death, which created a unique opportunity for cetuximab as a senolytic agent. Combination therapy reduced EGFR signaling and induced macrophage-mediated antibody-dependent cellular cytotoxicity, thereby increasing the anti-tumor therapeutic efficacy of OD-0J1.