Prediction of Therapeutic Potential of CD19 CAR-T Cells for LBCL By Histone Mark Analyses of Core Epigenetic Programming

• Published in: Blood 2024-11, Vol.144 (Supplement 1), p.2028-2028
• Main Authors: Fiorenza, Salvatore, Cao, Yue, Zheng, Ye, Purushe, Janaki, Bock, Tamara, Kimble, Erik L, Janssens, Derek, Kirchmeier, Delaney, Vinaud Hirayama, Alexandre V., Gauthier, Jordan, Maloney, David G., Riddell, Stanley R., Nguyen-Shaw, Akira, Henikoff, Steven, Yang, Jean, Turtle, Cameron J.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2024-200589

Chimeric antigen receptor-T cell (CAR-T) therapy has produced durable responses in a subset of patients with otherwise incurable B-lineage malignancies. Efforts are underway to identify genetic and epigenetic programs in CAR-T cells that influence product quality, thereby enabling development of next generation CAR-T products. The subset-of-origin (SOO) for CAR-T cell manufacturing and the effects of serial stimulation are key factors that might influence CAR-T quality. We hypothesized that serial stimulation might impart different effects on the epigenetic programming of CAR-T cells manufactured from distinct subsets; and that these epigenetic programs might associate with outcomes of clinical CAR-T therapy. We previously showed that sequencing of DNA bound to transcriptionally permissive (H3K4me2) and repressive (H3K27me3) histone methylation marks identifies numerous important differences in CAR-T cells from healthy donors (HD) and patients that were not apparent by RNA-seq. Here, we applied novel histone mark analyses to identify epigenetic programs that impact outcomes of large B cell lymphoma (LBCL) patients receiving CAR-T cell therapy. We first validated a novel in vitro model generating a roadmap of epigenetic and transcriptional programs during longitudinal serial stimulation of CAR T-cells manufactured from distinct SOO. H3K27me3 and H3K4me2 analyses and RNA-seq were performed on ex vivo isolated CD8+ Naïve (N), Central Memory (CM) and Effector Memory (EM) subsets from HD, on each SOO-derived CAR-T cell product after manufacturing, and prior to cycles of serial stimulation with a CD19+ lymphoma cell line. We first derived principal component analysis (PCA) plots from the 3 datasets (H3K4me2, H3K27me3, RNA-seq), each comprising 15 samples (3 SOO, 5 timepoints) from 7 HD. In PCA plots of histone mark data, we identified two epigenetic programmatic axes. One axis distinguished the SOO of CAR-T manufacturing. A second axis recapitulated the stimulation history. Importantly, clustering of samples showed histone mark analyses clearly distinguished not only each ex vivo isolated CD8+ T cell subset and CAR-T cell products manufactured from each of those subsets, but also identified each of the cycles of stimulation of each subset-derived CAR-T cell product. In contrast, when PCA plots were generated from RNA-seq data, SOO and stimulation history was indistinguishable after first stimulation. Thus, histone mark analyses enabled dissection of epigenetic p