Class I HDAC inhibition enhances the stem-like memory properties of CRISPR-engineered CAR T cells in neuroblastoma

Manufacturing chimeric antigen receptor (CAR) T cells with a stem cell memory phenotype can enhance their persistence in patients. Histone deacetylase inhibitors (HDACis) targeting class I HDACs promote chromatin remodeling in virally manufactured CAR T cells, leading to the activation of the Wnt pathway, and ultimately improve CAR T cell persistence. However, it is unknown whether the persistence of CRISPR-engineered CAR T cells can also be enhanced through such HDACi-mediated epigenetic modulation. CAR T cells engineered using CRISPR-Cas9 to integrate a CAR construct into the T cell receptor alpha constant (TRAC) were treated with various class- or isoform-selective HDACis, and phenotypic, functional, and metabolic changes were assessed in vitro and in a GD2+ neuroblastoma xenograft model. Compared to untreated GD2 TRAC-CAR T cells, chidamide and mocetinostat increased the stem cell memory marker expression (CD62L⁺/CCR7⁺/IL-7Rα⁺). Chidamide induced a fragmented mitochondrial morphology and, in vivo, enhanced the persistence and naive phenotype of the GD2 TRAC-CAR T cells without upregulating exhaustion markers (PD-1/LAG3). These findings suggest that in the management of treatment-resistant pediatric solid tumors, utilizing chidamide during the manufacturing of CRISPR-engineered CAR T cells may enhance their persistence while retaining a naive phenotype.