EXTH-96. CT-2A sublines with divergent immune phenotypes: therapeutic implications for polyamine blockade

Abstract Polyamines are small molecules that play a critical role in tumor progression and in the regulation of immune responses within the tumor microenvironment. Elevated levels of polyamines in cerebrospinal fluid and tumor tissue have been reported in multiple studies and are shown to correlate with glioma grade. These findings suggest that the polyamine metabolic pathway may represent a therapeutic target in gliomas. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, expected to exert antitumor effects through polyamine depletion. Although the impact of DFMO against glioblastoma as a monotherapy appears limited, opportunities for synergy with other therapies remain understudied. In this study, we evaluated the therapeutic efficacy of DFMO using both in vitro and in vivo experiments with a murine glioma model (CT-2A) and patient-derived xenograft (PDX) cell lines. Among the models tested, CT-2A cells exhibited the highest expression of ODC1 and demonstrated strong sensitivity to DFMO in vitro. We compared two CT-2A variants: a commercially available line responsive to immunotherapy, and a widely used subline resistant to PD-1 blockade. While DFMO did not improve survival in mice implanted with the immunotherapy-sensitive commercial CT-2A line, it extended median survival by 8 days in mice bearing tumors derived from the immunotherapy-resistant subline. Transcriptomic analysis revealed that the DFMO-responsive, immunotherapy-resistant tumors exhibited lower baseline expression of immune-related genes, including those involved in interferon-β, interferon-γ, and IL-12 signaling pathways, compared to the immunotherapy-sensitive tumors. The latter showed markedly higher expression of STAT1 and interferon-γ and were enriched for immune-related Gene Ontology terms. These findings suggest that polyamine-targeted therapy may be appropriate for gliomas with an immunosuppressive, “cold” phenotype. Ongoing RNA sequencing analyses aim to further elucidate the immunomodulatory mechanisms of DFMO, and other agents comprising an adaptive toolkit to optimize and sustain anti-tumoral immunity.