BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell stemness and exhaustion

Abstract

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of <i>PRDM1</i>, encoding the BLIMP1 transcription factor, defined highly potent <i>TCF7</i> [encoding T cell factor 1 (TCF1)]-expressing CD8⁺ CAR T cells, whereas enrichment of <i>HAVCR2</i> [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]-expressing CD8⁺ T cells with elevated <i>PRDM1</i> was associated with poor outcomes. <i>PRDM1</i> knockout promoted <i>TCF7</i>-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of <i>PRDM1</i> deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of <i>NR4A3</i> and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of <i>PRDM1</i> and <i>NR4A3</i> skewed CAR T cell phenotypes away from TIM-3⁺CD8⁺ and toward TCF1⁺CD8⁺ to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with <i>PRDM1</i> and <i>NR4A3</i> single knockout alone. These data underscore dual targeting of <i>PRDM1</i> and <i>NR4A3</i> as a promising approach to advance adoptive cell immuno-oncotherapy.