Uncovering Synergistic Pathways for T Cell Stemness and Improved Adoptive Cell Therapy Outcomes 2257598

Abstract Introduction Adoptive T cell therapy (ACT) has revolutionized cancer therapy with remarkable success in liquid cancers, but efficacy in solid tumors remains limited due to poor T cell infiltration and persistence. Stem cell memory T cells (Tscm) are a potent subset with enhanced self-renewal, persistence, and anti-tumor responsiveness. Skewing T cells towards a Tscm phenotype prior to transfer represents a promising strategy to improve ACT outcomes. While pathways, such as PI3K-AKT-mTOR and Wnt/β-catenin, are implicated in Tscm differentiation, the interactions between these complex pathways in regulating stemness and anti-tumor function remain poorly understood, representing a potential therapeutic target to improve efficacy. Methods This study evaluated whether combining small molecules targeting distinct pathways could synergistically enhance Tscm differentiation and anti-tumor efficacy. We used small molecule PI3K, AKT, and GSK3 inhibitors, known regulators of Tscm differentiation, either as single agents or in combination, to condition CD8+ OT-1 T cells prior to ACT. Cells were characterized by flow for stemness markers (e.g., TCF1, FOXO1, Slamf6) and exhaustion markers (e.g., PD-1, Tim-3, Lag-3). Conditioned T cells were adoptively transferred into tumor-bearing mice, and tumor size and survival were monitored. Results In vitro, dual treatments enhanced expression of Tscm-associated markers (TCF1, FOXO1, Slamf6) and reduced expression of PD-1, Tim-3, and Lag-3. In vivo, the combination of a PI3K inhibitor and a GSK3 inhibitor significantly reduced tumor size. Conclusion These findings suggest that the PI3K-AKT-mTOR and Wnt/β-catenin pathways interact to regulate Tscm differentiation, enabling enhanced anti-tumor activity. These findings provide valuable insights into the mechanisms driving stemness and persistence, offering a promising framework for advancing ACT strategies in melanoma and other cancers, with strong potential to improve clinical outcomes. Funding Source NIH grants: AI155015 and CA257954 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)