Acute myeloid leukemia (AML) is an aggressive blood cancer characterized by uncontrolled myeloid cell proliferation. Current curative therapy primarily relies on hematopoietic stem cell transplantation (HSCT) due to its graft-versus-leukemia (GVL) effect, mainly mediated by donor T cells. While T-cell based immunotherapies show promise, their application in AML remains limited. Innate immunity, particularly Natural Killer (NK) cells, is emerging as a significant player in tumor surveillance. NK cells recognize and kill tumor cells, particularly those downregulating MHC-I, a mechanism frequently used by tumors to evade T-cell mediated immune responses. This study utilizes Rag2⁻/⁻ mice, which lack functional T and B cells but have hyperactive NK cells, to investigate the role of NK cells in AML development. The researchers hypothesize that the hyperactive NK cells in these mice will significantly influence AML progression and will provide insights into NK cell-mediated immunotherapy strategies for AML.

The existing literature supports the role of both adaptive and innate immunity in AML. Allogeneic HSCT leverages the GVL effect, predominantly through donor T cells recognizing tumor-specific antigens presented by MHC-I. However, AML frequently evades T-cell surveillance through mechanisms like MHC-I downregulation. This makes NK cells particularly relevant, as they can recognize and kill cells lacking self-MHC-I. Previous studies have explored NK cell-based immunotherapies for AML, but further research is needed to understand the precise role of NK cells in different AML subtypes and their potential for therapeutic targeting. The use of Rag2⁻/⁻ mice, which provide a model with functional NK cells but lack adaptive immunity, is a novel approach to isolate and study the effects of NK cells on AML development.

The researchers established an MLL-AF9 AML mouse model by transducing MLL-AF9 into bone marrow progenitor cells and transplanting them into sublethally irradiated recipient mice (WT, Rag2⁻/⁻, and NSG). AML progression was monitored through survival curves, peripheral blood (PB), spleen, and bone marrow (BM) GFP+ cell counts. Gene expression profiling was performed on AML cells from different mouse groups using RNA-Seq. The role of MHC-I was investigated by targeting B2m using CRISPR/Cas9. NK cell numbers, activation markers (CD107a, CD69, Sca-1), and cytotoxicity were assessed by flow cytometry and co-culture assays. The effect of NK cell depletion was also evaluated using anti-NK1.1 antibody. Similar experiments were conducted with RUNX1-ETO9, rAM, and cSAM AML models. Serial transplantation of AML cells was used to generate more aggressive AML lines and evaluate changes in immunogenicity. Statistical analysis included Kaplan-Meier survival curves, Student's t-test, Mann-Whitney test, and one-way ANOVA.

The study found that AML development was significantly slower in Rag2⁻/⁻ mice compared to WT and NSG mice. This difference was attributed to the hyperactive NK cells present in Rag2⁻/⁻ mice. NK cells in Rag2⁻/⁻ mice showed increased numbers, higher expression of activation markers (CD107a, CD69, Sca-1), and enhanced cytotoxicity towards leukemia cells. Depletion of NK cells in Rag2⁻/⁻ mice reversed the protective effect, accelerating AML progression. B2m depletion, leading to MHC-I downregulation and NK cell activation, inhibited AML growth. RNA-Seq analysis revealed distinct gene expression profiles in AML cells from Rag2⁻/⁻ mice, with upregulation of genes related to innate immune and inflammatory responses. Interestingly, the immunogenicity of AML changed during tumor evolution, with serially transplanted, more aggressive AML lines becoming more susceptible to NK cell-mediated suppression. These findings were also observed in RUNX1-ETO9-induced AML, but not consistently in rAM and cSAM models, suggesting a degree of model-specific heterogeneity.

The study demonstrates a significant role for NK cells in controlling AML development, particularly in specific AML subtypes. The slower AML progression observed in Rag2⁻/⁻ mice directly links hyperactive NK cell activity to tumor suppression. The findings support the potential of NK cell-based immunotherapies for AML treatment. The observed shift in AML immunogenicity during tumor progression implies that NK cell targeting might be most effective against specific stages or subtypes of AML. The differential response observed in different AML models highlights the complexity of AML and the need for tailored therapeutic strategies based on the specific oncogenic drivers and immunological context. Future research should focus on identifying specific NK cell activating ligands on AML cells and developing targeted therapies to enhance NK cell activity against AML.

This research demonstrates the critical role of hyperactive NK cells in suppressing AML development in a Rag2⁻/⁻ mouse model. The study highlights the potential of NK cell-based immunotherapy for AML, particularly targeting specific AML subtypes or stages. Future studies should investigate the specific mechanisms underlying NK cell-mediated AML suppression and explore the development of targeted NK cell-based therapies.

The study primarily focused on MLL-AF9-induced AML and the results may not generalize to all AML subtypes. The use of mouse models limits the direct translatability to human AML. Further research is needed to validate these findings in human AML and to optimize NK cell-based therapeutic strategies for clinical application. The study also acknowledges some inconsistencies between different AML models, pointing to a level of model-dependent variation in the results.