Introduction

Acute myeloid leukemia (AML) is characterized by the aberrant proliferation of immature myeloid cells, resulting in bone marrow failure and high mortality risk. Despite advancements in conventional therapies, such as chemotherapy and hematopoietic stem cell transplantation (HSCT), AML still exhibits poor long-term survival rates, particularly among high-risk patients. The emergence of immunotherapy has revolutionized cancer treatment by providing innovative methods to target cancer cells while preserving healthy tissues. Natural killer (NK) cells, integral to innate immunity, have garnered significant interest in AML therapy due to their capacity to identify and eradicate malignant cells without prior sensitization. This review explores the intricate relationship between NK cells and AML, analyzing the dysregulation of NK cell function in patients with AML and examining innovative strategies to exploit NK cell-mediated cytotoxicity for therapeutic purposes. The review addresses challenges inherent in NK cell therapy, including AML cell evasion mechanisms and the imperative to optimize NK cell activation and persistence within the hostile tumor microenvironment. In addition, recent advancements in NK cell-based immunotherapies, ranging from adoptive NK cell transfer to chimeric antigen receptor (CAR)-NK cell engineering, are surveyed, with a discussion on their potential implications for AML treatment. Finally, the significance of ongoing research efforts in elucidating the mechanisms underlying NK cell dysfunction in AML is underscored, emphasizing the translation of these insights into effective therapeutic interventions to enhance outcomes for patients with AML.

Acute myeloid leukemia

AML is a hematologic malignancy marked by the accumulation of genetic abnormalities in hematopoietic stem/progenitor cells, impairing differentiation and causing dysregulated proliferation, resulting in autonomous and disordered proliferation of immature blasts (leukemic cells). Intensive chemotherapy is the primary treatment for patients with AML, with some undergoing HSCT based on risk stratification [1].

Recent years have seen the exploration of novel therapeutic options to enhance survival outcomes, including targeted gene and cytokine therapies, along with immunotherapies. Immunotherapy holds promise in leveraging the immune system of the patient to eliminate leukemia cells [2,3,4]. However, achieving better treatment outcomes in AML remains a significant challenge, requiring ongoing efforts.

Natural killer cells

NK cells, a subset of innate lymphoid cells, exhibit robust cytotoxic capabilities, enabling them to directly eliminate virus-infected or cancerous cells without prior activation or exposure to antigens [5, 6]. Unlike T cells, which may cause collateral damage to host tissues, NK cells undergo apoptosis following cytotoxic activity, rendering them potentially advantageous for therapeutic interventions [7].

NK cells express two distinct types of receptors on their surface [8]: activating receptors, including natural cytotoxicity receptors (NCRs), killer immunoglobulin-like receptors (KIRs), CD94/NK group 2C (NKG2C), NK group 2D (NKG2D), and DNAX accessory molecule-1 (DNAM-1) [9]; and inhibitory receptors, such as leukocyte immunoglobulin-like receptors (LIRs), inhibitory KIRs, and NK group 2A (NKG2A) [10, 11]. These receptors function through ligand recognition mechanisms.

In addition to their direct cytotoxic effects, NK cells facilitate antibody-dependent cellular cytotoxicity (ADCC) against tumor cells. This process is primarily mediated by the binding of CD16 (FcɤRIIIA) and CD32 (FcɤRIIC) receptors on NK cells to various antibodies. Upon engagement of Fc receptors, NK cell degranulation is triggered, leading to cell death [12]. Furthermore, NK cell activity is subject to modulation by other immune cells, such as activated dendritic cells and macrophages [13].

Several cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-β, released from tumor cells can impair NK cell function despite their role in overcoming tumor immune evasion [14, 15]. While the combination of IL-10 and IL-15 has been demonstrated to enhance NK cell cytotoxicity [16], the secretion of IL-10 by regulatory T cells (Tregs) may detrimentally affect NK cell activity [17].

In recent years, NK cells have gained attention due to significant advancements in immunotherapy and its clinical applications [18, 19]. The well-established relationship between tumors and NK cells is evidenced by preclinical studies indicating increased tumor growth rates in the absence of NK cells [23, 24]. Signals through MHC class I (MHC-I) inhibitory mechanisms inhibit the activation signals of NK cells. Downregulation of MHC-I renders tumor cells identifiable and susceptible to NK cell-mediated killing because activation signals override inhibitory signals [25].

Natural killer cells in acute myeloid leukemia

AML blasts are susceptible to attack by T lymphocytes and NK cells due to the expression of MHC class I molecules, leukemia-associated antigens (LAAs), or NK cell activation ligands [26]. However, AML can evade immunity through mechanisms that suppress the anti-leukemic functions of immune cells [27, 28].

Cell numbers

In patients with AML, NK cell numbers are the lowest at diagnosis and upon disease relapse. Conversely, they increase during remission [29, 30]. High reconstitution of NK cells after HSCT is associated with improved 2-year survival rates, reduced CMV reactivation rates, decreased risk of relapse, lower non-relapse mortality rates, and improved progression-free survival [31]. Decreased NK cell numbers on day 60 post-HSCT are associated with an increased risk of relapse [32].

Differentiation

NK cells differentiate from progenitors (CD34dim/CD117/NKG2A) into immature CD56+NK cells (CD34/CD117+/CD56±) and finally to functional CD56bright NK cells (CD34/CD117±/CD94+). Functional maturation occurs via the expression of NKG2A or KIRs [33]. Patients with a low-maturity profile of NK cells have inferior outcomes, including 3-year overall survival [34], suggesting the importance of NK cell maturation status in predicting long-term outcomes.

Downregulation of activating receptors

The expression of NCRs, such as NKp46, NKp44, and NKp30, is downregulated in NK cells of the majority of patients with AML [35]. In addition, reduced expression of DNAM-1 was observed in NK cells in patients with AML compared to that in controls [36]. Patients with AML with low NCR expression had shorter remission durations, whereas NCR expression was recovered in patients with AML who achieved CR [32], suggesting the importance of activating NK cell receptor expression in AML [37].

Upregulation of inhibitory receptors

The high expression of inhibitory receptors plays a crucial role in AML cell evasion and NK cell immunity [38, 39]. Some studies have reported that recipient leukocyte ligand and donor KIR mismatches lead to reduced relapse rates in patients with AML [40, 41]. Upregulation of CD94/NKG2A is associated with decreased cytotoxicity of NK cells against AML cells [42].

Immune checkpoint molecules

Immune checkpoint molecules, such as PD-1, T cell immunoglobulin, and the ITIM domain (TIGIT) regulate NK cell activity [43]. PD-L1 expression in patients with AML has been reported to modulate recognition by immune cells, correlating with favorable outcomes, such as decreased risk of relapse and extended overall survival [44]. TIGIT, which is expressed in NK cells, binds to CD112, CD113, and CD155 ligands such as DNAM-1. Reduced expression of DNAM-1 in patients with AML may promote binding to TIGIT ligands, sending inhibitory signals, and potentially contributing to tumor immune evasion. Blocking TIGIT enhances NK cell cytotoxicity [45].

In summary, various roles of NK cells in AML progression have been highlighted. Many studies have laid the groundwork for the development of various cell-based therapies targeting AML.

Natural killer cell therapy for acute myeloid leukemia

NK cell therapy is an immunotherapeutic option. In patients with AML, the function of NK cells is suppressed, allowing AML cells to evade immune cells. Numerous clinical trials of various NK cell-based immunotherapies are currently underway [2, 4]. An overview of NK cell therapy is illustrated in Fig. 1, and ongoing clinical trials are summarized in Table 1.

Fig. 1
figure 1

Natural killer cell therapy for acute myeloid leukemia. In patients with AML, downregulation of HLA-1 expression in AML cells and a decrease in the expression of activating receptors, such as NCR and DNAM-1, have been observed. In addition, NK cell function is suppressed by inhibitory receptors. CAR-NK cells targeting CD33 or CD123, as well as CIML-NK cells utilizing cytokines, have been developed. Furthermore, antibodies targeting ADCC activity and inhibitory receptors have been developed. DNAM-1, DNAX accessory molecule-1; NCR: natural cytotoxicity receptor; TIGIT, T cell immunoglobulin and ITIM domain; iKIR, inhibitory killer cell immunoglobulin-like receptor; NKG2A, NK group 2A; NKG2DL, NK group 2D ligand; HLA, human leukocyte antigen

Table 1 Ongoing clinical trials

Cell source

Autologous NK cells

Peripheral blood NK cells can be expanded ex vivo and reintroduced into the body as autologous NK cells [46]. Autologous NK cell therapy uses patient blood to minimize the risk of graft-versus-host disease (GVHD).

Injected cells can proliferate in vivo; however, in AML, significant therapeutic outcomes are often hindered by the inhibitory effects of human leukocyte antigen (HLA) ligands. In addition, autologous NK cells obtained from heavily pretreated patients may show reduced cytotoxic activity [47].

Allogeneic NK cells

Allogeneic NK cells were obtained from healthy HLA-matched or haploidentical donors, expanded ex vivo, and administered after removing T cells to prevent GVHD [48]. Donor-isolated NK cells show graft-versus-leukemia (GVL) effects against AML cells due to a KIR-KIR ligand (KIR-KIRL) mismatch [49]. Allogeneic NK cell therapy induced complete remission in five of 19 patients with AML with a poor prognosis. The remission rates were significantly higher when KIR ligand-mismatched donors were used [50]. Similar to autologous NK cells, challenges include timely ex vivo expansion, suboptimal activation to demonstrate clinical efficacy, and a lack of in vivo persistence [51].

Cytokines play an important role in NK cell function and have been used to overcome these challenges. IL-2 activates NK cell proliferation, and systemic administration after NK cell infusion may enhance the therapeutic efficacy [52].

The use of IL-15, which is less toxic upon systemic administration than IL-2, has also been investigated [53]. The IL-15 super-agonist complex, ALT-803, was identified as a safe agent in a Phase I trial targeting elderly patients with AML who relapsed after HSCT. Furthermore, NK cells expressing IL-15 or IL-12 can mitigate toxicity from systemic cytokine administration [54, 55].

Umbilical cord blood-derived NK cells

Umbilical cord blood (UCB) provides a readily available off-the-shelf allogeneic NK cell source. UCB-derived NK cells express CD3-CD56+ and are classified as undifferentiated CD56bright or mature CD56dim NK cells [56].

UCB-derived NK cell therapy, either as a maintenance therapy after chemotherapy or in combination with HSCT, has shown promising results [57]. Challenges include improving persistence and develo** strategies to maintain donor NK cell function in vivo [58, 59].

Cell line-derived NK cells

Over the past 20 years, eight cloned NK cell lines, namely, NK-92, YTS, and NKL have been established. Commonly used NK cell lines exhibit differences in phenotype and function [60]. NK-92 is the only cell line that has undergone clinical trials to evaluate safety and efficacy.

NK-92 cells can easily proliferate in vivo but lack many conventional NK cell markers and require irradiation before infusion to prevent uncontrolled proliferation. They demonstrate cytotoxicity against leukemia cells both in vitro and in vivo [61]. Although clinical trials have demonstrated the safety and tolerability of NK-92 cells, their efficacy has not met expectations [62]. NK-92 cells for refractory/relapsed AML did not show antitumor responses after infusion [63, 64]. We revealed that high expression of CD112 is associated with poor prognosis in patients with AML, and the introduction of DNAM-1 into NK-92 cells enhances their anti-AML effect [65].

NK cells derived from induced pluripotent stem cells (iPSCs) were used as standardized “off-the-shelf” products for all patients. iPSC-derived NK cells have been shown to suppress tumor growth in vivo in leukemia cell lines without exogenous IL-2 or IL-15 [66].

Cytokine-induced memory-like (CIML) NK cell

NK cells activated in response to interleukins, such as IL-12, IL-15, and IL-18, exhibit memory-like features and demonstrate long-term effector functions [67, 68]. No serious adverse events were observed, and clinical trials administering CIML NK cells in combination with chemotherapy to patients relapsing after HSCT are ongoing [69].

Chimeric antigen receptor (CAR)-NK cell

Since the introduction of CAR-T cell therapy, there has been significant interest in enhancing cytotoxic functions through genetic modifications [70, 71].

CAR-T therapy faces challenges, such as cytokine release syndrome, neurological toxicities, off-target effects, and manufacturing timelines.

In contrast, NK cells can avoid toxicity arising from nonspecific recognition of normal cells by binding to human leukocyte antigen (HLA) ligands on leukemia cells. NK cells recognize self and non-self-cells through various activating and inhibitory receptors on their surfaces [72, 73], which are known to not cause GVHD. In addition, they have a short lifespan, favorable cytotoxic profiles, and lower manufacturing costs, making them promising alternatives to T cells [74, 75].

Unmodified NK cell infusions have shown some efficacy in patients with AML, but their short lifespan and modest therapeutic outcomes have shifted the focus to CAR-NK cells, whose efficacy has been evaluated in various preclinical studies and clinical trials.

CAR-NK cells incorporating signaling domains, such as 2B4 or 4-1BB, have demonstrated anti-AML cytotoxic activity both in vitro and in vivo [76]. CAR-NK-92 targeting CD123 is highly expressed in AML cells and is engineered to express human IL-15, which has enhanced in vivo persistence [77]. Other CAR-NK cells targeting CD4 [78], CD276 [79], or NKG2D [80], and attempts to enhance cytotoxic activity by knocking out inhibitory NK cell receptors, such as NKG2A or TIGIT, are underway.

The first CAR-NK cell therapy was reported in 2018, which evaluated the safety and efficacy of third-generation CD33 CAR-NK cells derived from the NK-92 cell line with CD28 and 4-1BB costimulatory domains in three relapsed/refractory (R/R) patients with AML. Although CAR-NK-92 cells can be produced at much lower costs than CAR-T cells, the significant reduction of CAR-NK cell numbers within a week post-infusion due to pre-infusion irradiation and the lack of sustained remission remains a challenge [81].

However, CAR-NK cells have several drawbacks. First, NK cell numbers are limited and their lifespan is short (approximately 1 to 4 weeks). Incorporation of cytokines, such as IL-2 or IL-15, into CAR-NK cells may improve their proliferation and persistence [74]. Various methods, including the administration of cytokines ex vivo or the use of feeder cells, have been used to enhance proliferation [82]. Second, there are no specific optimal targets. Some surface markers of AML cells are shared with hematopoietic stem cells, making it crucial to identify the optimal leukemia-specific markers that can be specifically targeted by CAR-NK cells [83, 84]. In addition, antigen downregulation or loss by AML cells may occur, leading to the development of CAR-NK cells that target the two antigens to overcome this challenge [85]. Although CAR-NK cells typically function only when antigens are expressed on the cell surface, CAR-NK cells that recognize intracellular antigens presented by MHC molecules via TCR have also been developed [86]. Finally, there is a high risk of apoptosis and difficulty in genetic manipulation. It is necessary to optimize methods such as electroporation and the viral vectors used for genetic manipulation [87, 88].

Antibodies

Antibodies targeting tumor-associated antigens

Antibodies targeting tumor-associated antigens play a significant role in inducing ADCC in NK cells. Preclinical studies have investigated Fc-optimized antibodies against various potential antigens, such as IL-1 receptor accessory protein (IL1RAP), CD33, and CD133 [89,90,91]. Furthermore, novel therapies that combine NK cell therapy have shown promising results [92, 93].

Antibodies targeting NK cell inhibitory receptors

Inhibitory receptors on NK cells play a crucial role in immune evasion by tumor cells, making them excellent targets for cancer immunotherapy. In addition to MHC-I inhibitory leukocyte immunoglobulin-like receptors (LIR), KIR, and CD94/NKG2A receptors, other NK cell inhibitory receptors have been implicated in cellular dysfunction [94]. CD200R is also known to be an inhibitory receptor for NK cells. Overexpression of CD200 suppresses the antitumor activity of NK cells in patients with AML, leading to an increased risk of relapse; however, inhibition of CD200 has been reported to restore NK cell function [95].

Targeting the KIR2DL1/2/3 NK inhibitory receptors with IPH2101 has shown efficacy both in vivo and in clinical trials [96]. However, no significant improvements were observed in median survival or response rates. Combinations of anti-NKG2A, anti-KIR, and anti-LIR-1 antibodies may be necessary to enhance the therapeutic efficacy [97, 98].

Immune checkpoint inhibitors

Like T cells, NK cells express cytotoxic T lymphocyte-associated protein 4 (CTLA4), LAG3, and PD-1, suggesting that immune checkpoint receptors regulate NK cell activity [99]. TIGIT and CD96 (TACTILE) as well as T cell immunoglobulin and mucin domain 3 (TIM-3) have also been identified as inhibitory receptors expressed on NK cells [100].

In clinical trials, monotherapy with anti-PD-1 antibodies for AML did not show significant efficacy [101]. However, ipilimumab treatment after allogeneic HSCT resulted in favorable responses in 22 patients with various hematological malignancies, including 12 patients with AML [102]. TIM-3 is a co-inhibitory receptor that recognizes galectin-9 as its ligand. Blocking TIM-3 has been shown to restore NK cell function [103, 104], and the efficacy of MBG453 (a TIM-3 antibody), alone or in combination with other agents, has been analyzed.

Clinical trials are also being conducted on anti-LAG3 antibodies and anti-TIGIT therapies among others.

Challenges and prospects

Our understanding of the role of NK cells in leukemia and their function in anti-leukemia therapy has improved dramatically. Patients with leukemia often experience NK cell dysfunction or a reduced number of NK cells. NK cell infusion is beneficial in temporarily restoring these functions. Compared with T cell-targeted therapies, which are costly to manufacture and have lower safety profiles, NK cells are gaining attention in immunotherapy because of their lower neurotoxicity and incidence of cytokine release syndrome in CAR-T therapy.

NK cells represent a promising immunotherapy for rapid tumor detection, antitumor effects, and the ability to enhance immune responses. Despite numerous immune evasion mechanisms, the crucial role of NK cells in AML immune surveillance has been demonstrated. From the adoptive transfer of NK cells and cytokines to immunomodulatory agents, antibodies, and gene editing, NK cells have paved the way for better management of AML.

Several clinical trials have investigated the potential of NK cell infusions in combination with conventional therapies. NK cell therapy offers advantages over other cell therapies, including the recognition of tumor cells independent of MHC, the potential to treat a broader population, ease of expansion in vivo, and cost-effective manufacturing of off-the-shelf products tailored to individual patients.

However, several challenges remain in enhancing the potential of NK cells in AML immunotherapy. These include blocking inhibitory receptors, eliminating Treg activity, and neutralizing immunosuppressive cytokines. Providing the essential cytokines and growth factors necessary for NK cell activation, proliferation, and survival is challenging.

The majority of these studies are still in the preclinical stage, highlighting the need for further research and a deeper understanding of the mechanisms of NK cell dysfunction and immunotherapy-based approaches in AML to validate and implement these therapeutic strategies.

Conclusion

Despite advances in chemotherapy, AML remains a challenging hematologic malignancy to cure. NK cells have shown potent anti-AML effects without causing adverse events, such as GVHD, neurotoxicity, or cytokine release syndrome. However, the effector functions of NK cells are inhibited in immunosuppressive microenvironments. Functional impairment of NK cells by the heterogeneous TME poses a significant obstacle to maintaining anti-AML activity. Achieving desirable outcomes with NK cell therapy requires enhancing NK cell cytotoxicity, improving the recognition of target cells, and maintaining cytotoxic functions. Thus, NK cell therapy may be a viable treatment option for AML.