Natural killer cell heterogeneity reflects AML cell-states and predicts clinical outcome at diagnosis

Abstract Background: Natural killer (NK) cells are innate lymphocytes with the unique ability to eliminate transformed or infected cells without prior sensitization. Their differentiation in the bone marrow follows a continuum from CD56Hi immature NK cells, which secrete inflammatory cytokines, to CD56Dim mature NK cells, which possess potent cytolytic capacity. This functional versatility has positioned NK cells as attractive candidates for immunotherapeutic strategies in cancer, including acute myeloid leukemia (AML). AML is a genetically and phenotypically heterogeneous hematologic malignancy defined by clonal expansion of immature myeloid blasts that disrupt normal hematopoiesis. Although advances in NK cell-based therapies, including cytokine-induced memory-like NK cells, bi- and tri-specific antibodies, and chimeric antigen receptor (CAR)-NK approaches, have been made, but their effectiveness remains limited. The complex immunosuppressive environment and diversity of AML blast subtypes likely contribute to this limitation. A deeper understanding of the impact of AML heterogeneity on NK cell states is crucial for refining therapeutic strategies aimed at treating AML. Results: We performed high-dimensional 20-color flow cytometry on bone marrow samples from >50 newly diagnosed AML patients, alongside healthy controls, to comprehensively profile both leukemic blasts and NK cell subsets. As expected, NK, T, and B cells were markedly reduced in AML. We identified distinct blast subtypes, including monocytic, myelomonocytic, progenitor-like, and stem-like, and then mapped NK cell phenotypes across these AML subtypes. Compared to healthy donors, AML samples exhibited a significant loss of mature CD56Dim NK cells, suggesting a block in NK cell maturation. Notably, NK cells in AML expressed canonical markers shared with myeloid cells, indicating leukemic perturbations. These NK cells also exhibited altered cytolytic functions, consistent with their dysfunction.AML subtype analysis revealed that monocytic AML was enriched for the most dysfunctional NK subsets. This reprogramming suggests potential therapeutic leverage that could be utilized to enhance treatment efficacy by targeting dysfunctional NK cells in AML. To further characterize NK cell heterogeneity, single-cell transcriptomic analysis was performed on enriched NK cells from AML patients at the time of diagnosis. K-means clustering identified seven transcriptionally distinct NK cell states. Patients with enrichment of NK cells mimicking the healthy NK subset (NK4) consistently achieved durable remissions, while those with predominant perturbation of transcriptome subsets (NK2,3,5) experienced early relapse. Further analysis of relapse-associated clusters revealed upregulation of c-Jun N-terminal kinase (JNK) signaling pathways that impair NK cytotoxic function. The observed upregulation of the JNK pathway identifies a potential therapeutic target, as several JNK inhibitors are available. These findings support the rationale for conducting trials that evaluate JNK inhibitors to restore NK function and improve patient outcomes. Based on these data, an NK cell gene signature was derived to predict clinical outcome at the time of diagnosis. This signature was validated at the protein level in an independent cohort of >100 AML patients using flow cytometry and achieved >80% accuracy in distinguishing remission from relapse, thus making it a reliable therapeutic biomarker. Conclusion: Our study reveals that AML-driven NK cell reprogramming is linked to the leukemic cell state and decisively determines therapeutic response. The integration of flow cytometry with single-cell transcriptomics uncovers NK cell phenotypes that mirror the AML environment and predict patient outcomes. Collectively, these results establish NK profiling as a practical biomarker and a powerful tool for guiding patient selection and care, marking a pivotal advance toward precision NK cell–based immunotherapies. In contrast to traditional approaches of immunotherapy, our stratified methodology ensures that each patient receives the right therapy tailored to their specific NK and AML phenotype.