Natural Killer (NK) cell-based immunotherapies are often limited by rapid exhaustion and apoptotic depletion within the hostile tumor microenvironment (TME). Through comprehensive pro-apoptotic profiling, we identified that elevated expression of both BIM and NOXA in NK cells serves as a robust prognostic indicator of poor clinical outcomes across various malignancies, including pancreatic adenocarcinoma. Our findings reveal that both BIM and NOXA are markedly upregulated in human NK cells upon exposure to TME-derived suppressive cues, such as IL-6 and TGF-β1, collectively driving immune vulnerability and functional impairment.
To counteract these apoptotic pressures, we utilized CRISPR-Cas9 RNP complexes to disrupt the BIM or NOXA locus in primary human NK cells. Despite the involvement of both genes in NK dysfunction, genetic ablation of NOXA alone significantly augmented cytotoxic resilience under stringent conditions—including long-term serial killing challenges and patient-derived organoid models—yielding functional gains that were notably superior to those observed in BIM-deficient counterparts. Furthermore, NOXA-KO NK cells exhibited a synergistic response to IL-15, maintaining proliferative vigor and effector functions beyond the levels achieved by cytokine stimulation alone. This enhanced performance was metabolically supported by a simultaneous increase in oxygen consumption rate (OCR) and oxidative phosphorylation (OXPHOS). Collectively, these results establish NOXA as a pivotal therapeutic target, providing a robust framework for engineering next-generation NK cells with sustained persistence and metabolic adaptability for solid tumor immunotherapy.