MLL4-Menin Complex Inhibition Promotes Central Memory In CD8 CAR-T Cells

Abstract

CAR-T cell immunotherapy is a highly efficacious treatment for CD19-positive hematological malignancies, however, some patients are non-responsive for reasons that are not well understood. Clinical efficacy has been correlated with long-term persistence, a propensity that can be predicted by the differentiation state of transplanted cells. Despite this, decades-old methods for expanding T cells have not been updated to prevent the deleterious effects of excessive differentiation in CAR-T cells. Uncoupling proliferation and differentiation is a long-held goal in the field of immunotherapy with both cytokines and pharmacological approaches being implemented to dissociate these parallel processes. Histone methyltransferases rewire transcriptional programs in T cells and simultaneously regulate multitudes of genes, making them attractive targets for modifying the proliferation-differentiation axis. Despite this, only a handful of studies have examined their role in regulating the transcriptional programs of human CD8+ T cells. MLL4 (encoded by KMT2B) belongs to the six-member group of MLL histone methyltransferases. MLL1, a paralog of MLL4, has been implicated in regulating the maintenance of IL-4 and GATA-3 expression in TH2 CD4 memory T cell populations, however the function of MLL4 in human CD8+ T cells is unknown. We report a critical role for MLL4 in the proliferation and differentiation of CD8+ T cells. CRISPR-Cas9-editing of MLL4 uncoupled the processes of proliferation and differentiation, increasing proliferation but maintaining central memory T cell (TCM)-like populations, allowing for the production of increased numbers of TCM-like CD62L+CD45RO+ cells. Pharmacologically inhibiting the MLL4-Menin complex with MI-2 during T cell expansion enriched the frequency of minimally differentiated TCM-like CD8+ T cells. TCM-associated CD62L, CCR7, CD122 and CD127 surface markers were upregulated and early memory-associated transcription factor TCF7, LEF1, EOMES, and FOXP1 transcripts were increased. CD8+ CAR-T cells expanded in the presence of MI-2 responded earlier, while improving both tumor burden and survival in a NSG xenograft model of human leukemia. This finding has important translational impact in improving the persistence and proliferative capacity of CD8+ CAR-T cells.