JAG1 Blockade with oHSV Polarizes Macrophages to Create a Unique Vulnerability for Cetuximab-Mediated Senolysis of Glioma Cells

Kimberly Ann Rivera Caraballo, BSc (Advisors: Balveen Kaur, PhD and Ji Young Yoo, PhD)

          Oncolytic HSV-1 (oHSV) treatment induces Notch signaling and myelosuppression in the tumor microenvironment (TME) of preclinical models. In the clinic, upregulation of JAG1 gene expression was observed in recurrent high-grade glioma patients treated with CAN-3110. Since JAG1 is expressed on both glioma cells and tumor-associated macrophages (TAMs), and its expression correlated with a worse prognosis, we engineered a JAG1-antagonizing oHSV (OncoD-0J1) and interrogated its impact on the tumor and myeloid TME. OncoD-0J1 antagonized JAG1-mediated Notch signaling and had a significant therapeutic advantage in vivo, which relied on Notch signature in tumor cells. Kinome profiling revealed that OncoD-0J1 treatment suppressed CDK1, resulting in a G2/M cell cycle checkpoint as seen by cell cycle analysis. Cell cycle arrest led to senescence and correlated with increased reactive oxygen species, p62 accumulation, autophagosome accumulation, and senescence-associated beta-galactosidase activity. This resulted in increased production of inflammatory chemokines and DAMPs such as IL-1β and extracellular ATP. RNA sequencing of murine macrophages co-cultured with infected human tumor cells showed enrichment of chemotactic and pro-inflammatory pathways as well as increased Fc receptor activation following OncoD-0J1 infection. Single-cell RNA sequencing and flow cytometric analysis of F4/80⁺ cells isolated from infected tumors showed a shift from tumor-supporting TAMs to inflammatory macrophages upon OncoD-0J1 treatment. Senescent cells showed heightened EGFR activation as a mechanism to escape death, which created a unique vulnerability 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 response of OncoD-0J1 as a monotherapy.

Advisory Committee:

• Balveen Kaur, PhD, Chair
• Ji Young Yoo, PhD, Co-Chair
• Leomar Ballester, MD, PhD
• Jian Hu, PhD
• Jason Huse, MD, PhD

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