TRAIL orchestrates ThINKK-induced NK cell cytotoxicity against childhood acute lymphoblastic leukemia

Therapeutic Inducers of Natural Killer cell Killing (ThINKK) represent a novel class of immunotherapy designed to enhance the graft-versus-leukemia effect of hematopoietic stem cell transplantation in pediatric patients with high-risk or relapse leukemia. Our previous work identified high expression of TRAIL as a key signature of Natural Killer (NK) cell stimulation by ThINKK. In this study, we aim to elucidate the mechanisms underlying acute lymphoblastic leukemia (ALL) killing by ThINNK-stimulated NK cells. We performed NK cell cytotoxic assays using a panel of genetically diverse B-cell precursor ALL (BCP-ALL) cell lines and patients’ samples. We performed gene deletion and gene enforced expression in sensitive or resistant cell lines to investigate the role of TRAIL-receptors expression and death receptor-signaling pathway in BCP-ALL cell death induced by ThINKK-stimulated NK cells. These findings were further validated through the analysis of primary patients’ samples and transcriptomic profiling of a cohort of 320 BCP-ALL patients from the CHU Sainte-Justine. We found that BCP‑ALL with diverse genetic backgrounds are sensitive to ThINKK‑stimulated NK‑cell killing. In addition, our data revealed the dual role of TRAIL: a strong NK cell-activating receptor that induced rapid killing of TRAIL-R2-expressing BCP-ALL through cytolytic granule release and cytokine secretion, and then, a death receptor–ligand inducing BCP-ALL apoptosis following sustained engagement with its receptors. The transcriptomic analysis of BCP-ALL patients’ samples indicated that TRAIL-R2 and TRAIL-R1 are widely expressed across BCP-ALL subtypes and are not downregulated at relapse. These findings establish a mechanistic framework to guide patient stratification and therapeutic optimization.