Abstract 3094: Aurora-A inhibition sensitizes HPV-driven cancers to anti-CTLA-4 immunotherapy by modulating the tumor immune microenvironment

Abstract About 5% of cancers are caused by human papillomavirus (HPV), most of which are resistant to immune checkpoint inhibitors (ICIs). Alisertib (Ali), an Aurora kinase A inhibitor, may sensitize these cancers to ICIs by inducing apoptosis and DNA damage. This study investigates combining Ali with ICIs to enhance antitumor immunity and overcome resistance. We assessed Ali’s effects on apoptosis, DNA damage, and immunogenic cell death in HPV-positive cancer cell lines. Ali was tested alone or with ICIs in the mEER model, a surrogate for HPV+ oropharyngeal cancers. Changes in the tumor immune microenvironment (TIME) were analyzed by flow cytometry. RNA-seq evaluated gene expression across four groups: control, Ali, anti-CTLA-4, and combination. Gene set enrichment and Gene Ontology (GO) analyses identified key biological processes.Combination therapy with Ali and anti-CTLA-4 significantly reduced tumor size and improved survival, with 75% of treated mice surviving at 60 days compared to 0% of controls. Ali induced increased cleaved caspase-3 and PARP expression in tumor tissues, indicating enhanced cancer cell apoptosis. Flow cytometry showed enhanced T-cell polyfunctionality, with increased IFN-γ and granzyme production in tumor-infiltrating lymphocytes, along with reduced T-cell exhaustion (e.g., LAG3). A significant reduction in the Gr-1low Ly6G+ granulocytic subset of myeloid-derived suppressor cells (MDSCs) suggested an improved immune microenvironment. RNA-seq identified 22, 059 protein-coding genes with ≥1.5-fold increase compared to controls. Of these, 400 genes were upregulated by Ali, 3 by anti-CTLA-4, and 640 by the combination, with 191 genes upregulated in both Ali and combination groups. GO analysis revealed that shared genes activated immune-related processes like cell surface receptor signaling, macrophage activation, and T-cell cytokine regulation. In the Ali group, immune activation was linked to cytokine production and immune effector responses, while the combination showed enrichment for metabolic processes, suggesting metabolic reprogramming as a mechanism for enhanced immune responses and efficacy. The combination therapy increased CD8+ central memory T-cells, CD4+ memory T-cells, and elevated naive CD4+ T-cells, indicating enhanced T-cell activation and recruitment to the tumor, correlating with increased immune cell infiltration and improved survival. The combination of Ali and anti-CTLA-4 enhances outcomes in an HPV-driven murine model by boosting immune activation and reshaping the TIME. RNA-seq analysis shows that Ali drives immune responses, further amplified by anti-CTLA-4, promoting both immune and metabolic reprogramming. These findings suggest that combining Ali with ICI could overcome resistance and improve therapeutic outcomes in HPV-positive cancers. Citation Format: Soma Ghosh, Madison P. O’Hara, Tuhina Mazumdar, Lacin Yapindi, Li Shen, Jing Wang, Mitchell J. Frederick, Jagannadha K. Sastry, Faye M. Johnson. Aurora-A inhibition sensitizes HPV-driven cancers to anti-CTLA-4 immunotherapy by modulating the tumor immune microenvironment [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 3094.