Metabolically supercharged NK cells engineered with adenoviral E4ORF-1

Background: Chimeric antigen receptor (CAR) T and natural killer (NK) cells have achieved success in hematologic malignancies but show limited efficacy in solid tumors. A major barrier is the metabolically hostile solid tumor microenvironment (TME), where hypoxia, acidosis, and nutrient deprivation impair immune cell fitness, cytotoxicity, and persistence. Current metabolic engineering strategies that enhance single nutrient uptake or target one metabolic pathway provide only partial benefit and remain vulnerable to tumor metabolic plasticity. A critical unmet need is the development of immune cells with metabolic flexibility rather than single-pathway dependence. To address this, we explored a strategy inspired by viral metabolic rewiring. During adenoviral infection, the viral protein E4ORF-1 activates PI3K-AKT signaling, stabilizes MYC, augments nutrient uptake, and enhances glycolysis, oxidative phosphorylation (OXPHOS), and fatty acid oxidation (FAO). We hypothesized that engineering E4ORF-1 into NK cells would generate a viral-like metabolic state capable of withstanding nutrient restriction in solid tumors. Methods: Metabolic characterization included mitochondrial mass and membrane potential, Seahorse assays, and SCENITH. CAR-NK cells were cocultured with solid tumor cell lines, and cytotoxicity was quantified using xCelligence and IncuCyte platforms. In vivo efficacy was tested in xenograft models of hematologic (MOLM14) and solid tumors (SKOV3). Mechanistic studies incorporated bulk RNAseq, CyTOF profiling, and CRISPR-Cas9 knockout of AMPK. Results: E4ORF-1 expression significantly enhanced NK cell antitumor function across tumor models and sustained cytotoxicity and metabolic fitness under glucose-, glutamine-, or lipid-limited conditions. Western blotting confirmed coordinated upregulation of nutrient transporters and metabolic enzymes across glycolysis, OXPHOS, and FAO. E4ORF-1 NK cells also maintained a cytotoxic advantage under targeted metabolic inhibition; despite blockade of glycolysis, OXPHOS, FAO, or glutamine metabolism, they consistently demonstrated superior tumor killing, underscoring enhanced metabolic adaptability. Transcriptomic profiling showed preserved cytokine signaling and metabolic signatures even under nutrient restriction or after tumor challenge. Mechanistic studies identified AMPK as a central metabolic integrator required for the E4ORF-1 phenotype, as CRISPR deletion of AMPK abrogated metabolic and functional advantages. Incorporation of E4ORF-1 into CAR-NK cells improved tumor control and survival in vivo. Conclusion: E4ORF-1 enhances NK cell metabolic flexibility, enabling sustained antitumor activity and improving therapeutic potential for solid tumors. These findings establish viral gene-mediated metabolic rewiring as a promising platform to strengthen CAR-NK cell fitness and overcome nutrient competition in the TME.