DUSP6 ablation restores CAR T fitness impaired by tumor CD58 loss through invigoration of AP-1 signaling

Primary resistance to chimeric antigen receptor (CAR) T-cell therapies limited its widespread applications. Our prior genome-wide CRISPR/Cas9 screening revealed loss of CD58 as a crucial tumor intrinsic resistance factor by conferring insufficient immune synapse formation, impairing CAR-T activation and cytotoxicity. However, the specific signaling pathway and transcriptional changes that conveyed CAR T-cell dysfunction haven’t been addressed. Here we recognized an AP-1 dominated activation attenuation in CAR T cells impaired by tumor CD58 loss, driving synchronous mitochondrial biogenesis decrease, metabolic kinetics impairment, mitochondrial membrane potential loss and ROS accumulation. Moreover, this AP-1 attenuation triggered a death-receptor independent apoptosis through the intrinsic mitochondrial pathway. In seeking therapeutic strategies, we pharmacologically and genetically blocked three distinct inhibitory phosphatases positioned upstream of AP-1 signaling. Multi-faceted validation demonstrated dual specificity phosphatase 6 (DUSP6) blockade as an effective approach to supplement AP-1 signaling, notably decreased CAR T apoptosis, enhanced mitochondrial fitness, proliferation and long-term cytotoxicity. Transcriptomic profiles of DUSP6 ablated CAR T cells showed notably upregulated T-cell activation signatures and enriched metabolic pathways. Clinically, bulk and single-cell RNA-seq analysis revealed downregulated DUSP6 in patients responding to T cell-based immunotherapy, implying its relevance to outcome. Our findings repositioned CD58 not merely as an immune synapse component but as a metabolic checkpoint in CAR T biology, whose loss would trigger an AP-1-dependent mitochondrial derangement and created a permissive landscape for intrinsic apoptosis, ameliorable by ablation of inhibitory phosphatase DUSP6. Crucially, DUSP6 ablation presented a promising engineering target to potentiate CAR T-cell efficacy in broader applications.