1132 Enhancing melanoma therapy efficacy by protecting EZH2 activity in T cells

<h3>Background</h3> Metastatic melanoma treatment has been transformed by immune checkpoint blockade (ICB), yet only about half of patients respond effectively. The solid tumor microenvironment (TME) remains a major hurdle for T cell-based therapies, as tumor infiltrating lymphocytes (TILs) and cancer cells compete for metabolic resources, leading to T cell dysfunction and exhaustion. There is a critical need for new strategies to engineer adoptive cell therapy (ACT) that can maintain function in the challenging TME, especially if these strategies can be combined with other therapies to enhance clinical outcomes. Recent studies highlight the role of the histone methyltransferase EZH2 in protecting TILs from TME-induced exhaustion. EZH2, the catalytic part of the Polycomb Repressive Complex 2 (PRC2), acts as a transcriptional repressor by methylating lysine 27 of Histone H3 (H3K27me3). <h3>Methods</h3> To investigate the effect of EZH2 on T cells, we developed retroviral and lentiviral particles with wild-type EZH2 and various gain-of-function (GoF) EZH2 mutations. These viral particles were transduced into T cells to study their impact on T cell killing and responses to EZH2 inhibitors. Functional characterization of these mutant T cells was conducted using live cell imaging over a 72-hour period with an ImageExpress confocal HT.AI microscope. Changes in T cell biology following exogenous EZH2 expression were analyzed using immunoblotting and flow cytometry. <h3>Results</h3> Preliminary findings suggest that engineering T cells with cancer-evolved GoF EZH2 mutations prevents dysfunction and exhaustion within the TME. Additionally, these GoF EZH2 mutations, combined with a drug resistance mutation in T cells, help maintain EZH2 methyltransferase activity even in the presence of EZH2 inhibitors. This supports the hypothesis that engineering EZH2 mutations into T cells could create a more durable ACT, potentially usable in combination with EZH2 inhibitors to treat melanoma. <h3>Conclusions</h3> The metabolically stressful solid TME poses a significant challenge for successful ACT implementation. We explored enhancing T cells with improved EZH2 activity to better persist and infiltrate nutrient-depleted environments. EZH2 is crucial for melanoma cell survival by promoting proliferation, epithelial-mesenchymal transition, and invasion. While EZH2 inhibitors show promise as single agents and in combination with immune-based therapies for treating neoplasms, they also target T cells, causing exhaustion and limiting their clinical utility. Our preliminary data indicates that enhancing T cells with protected EZH2 function can significantly improve their persistence in melanoma treatment.