Even with advances in understanding the physiopathology of Acute Myeloid Leukemia (AML), most patients still relapse leading to poor long-term prognosis and cure rates. Natural Killer (NK) cells are regulated by opposing signals from receptors that activate and inhibit effector function and are known to mediate anti-leukemic immunosurveillance in AML, but the mechanisms by which they control hematopoietic neoplasms remain unclear.

We hypothesize that functionally impaired NK cells can predict poor outcomes in AML.

We analyzed the expression of DNAM-1 and NKG2D (activating) and NKG2A and KIR2DL1 (inhibitory) receptors on CD56+, CD56bright CD16- and CD56dim CD16+NK subsets, by flow cytometry, in 100 de novo AML bone marrow (BM), followed up from 02/2016 to 06/2022, 17 healthy BM (HBM) and 6 BM from remission after induction chemotherapy (AML-CR). Correlations with ELN2017 risk, complete remission (CR), relapse (R), measurable residual disease (MRD), overall survival (OS), and relapse-free survival (RFS) were verified. Co-culture of AML-exposed NK cells and K562 cell line was used to assay for killing and degranulation. Proteomics and genomics were accessed by CITE-seq single-cell sequencing.

All NK cell subsets were decreased in AML (p < .01). AML CD56+ NK cells showed decreased NKG2D (p <.01) and increased NKG2A (p = .04) and KIR2DL1 (p < .01), recovering to near healthy values in AML-CR. NKG2D and DNAM-1 activating receptors were decreased (p < .01), while inhibitory NKG2A (p = .02) and KIR2DL1 (p < .01) were increased in CD56dim NK (Most cytotoxic subset). Both activating receptors within CD56dim NK were decreased in ELN2017 adverse risk (NKG2D, p =.05; DNAM-1, p < .01), in relapse (p < .01), and in MRD+ patients after remission induction. Inhibitory receptors were increased in adverse risk (p < .01), relapse (NKG2A, p < .01; KIRDL1, p < .05), and MRD+ (NKG2A, p = .03) patients. Degranulation (p < .01) and target cell death (5:1, p = .01; 10:1, p = .002) were decreased in AML. Patients with lower frequency of CD56dim NK expressing low NKG2D and DNAM-1 showed less degranulation and target cell death (p = .03), whereas in those with higher inhibitory receptors lower degranulation (NKG2A, p = .06) and cytotoxicity (KIR2DL1, p = .005) was found. Both OS and RFS were decreased in patients with lower CD56dim NK (OS, p = .04; RFS, p = .03) and patients with lower expression of NKG2D (OS, p = .03; RFS, p = .008) and DNAM-1 (OS, p = .02; RFS, p = .11). Our single cell preliminary analysis showed that NK cells from AML display a distinct pro-inflammatory gene signature compared to HBM, which is strongly correlated to its impaired function.

Our data points that CD56dim NK shows an exhaustion phenotype with high expression of inhibitory and low expression of activating receptors. This imbalance leads to impaired cytotoxicity, compromised anti-leukemic activity, and can predict worst response to treatment, relapse, and MRD positivity, and so, can be considered an adverse risk prognostic factor associated with lower survival rates. On top of that, identifying a pro-inflammatory gene signature on AML NK cells can lead to the identification of pathways that prevent their activation and functionality, making it possible to intervene and prevent the onset of the disease. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy.