Biochemical mechanisms implemented by human acute myeloid leukemia cells to suppress host immune surveillance

Abstract

Acute myeloid leukaemia (AML) is a blood/bone marrow cancer originating from myeloid cell precusors capable of self-renewing. AML cells implement biochemical mechanisms which allow them not only to survive, but also to successfully escape immune surveillance. ln this work, we discuss crucial molecular mechanisms used by human AML cells in order to evade immune attack.

Human malignant tumors have developed a variety of effective molecular strategies that allow them to escape host immune surveillance, leading to disease progression. These tumors include hematological malignancies such as acute myeloid leukemia (AML), a blood/bone marrow cancer that originates from self-renewing myeloid cell precursors and rapidly becomes systemic. AML cells are capable of escaping immune attack, even though they are permanently exposed to host immune cells, including cytotoxic T cells (CTCs) and natural killer (NK) cells.¹ AML cells successfully implement biochemical mechanisms that allow them to inactivate cytotoxic lymphoid cells (NK cells and CTCs) both upon direct contact and at a distance.² In this case, they not only “fight back” against immune cells but also effectively prevent the actual process of cytotoxic immune attack. In this work, we discuss several important biochemical mechanisms that allow AML cells to form immunological synapses with cytotoxic lymphoid cells and comprehensively inactivate anti-cancer immunity at a distance.

T helper (Th)-type cells generate and secrete interleukin-2 (IL-2), a stimulatory cytokine that triggers the activation of NK cells and CTCs.³ Upon activation, these cytotoxic lymphoid cells become capable of attacking malignant (e.g., AML) cells delivering the proteolytic enzyme granzyme B to them. Granzyme B itself can directly activate one of the key apoptotic enzymes, caspase-3. However, granzyme B performs cleavage of the pro-apoptotic protein Bid, forming its active form tBid, which negatively impacts mitochondrial function and induces the release of cytochrome c, one of the major components of the electron respiratory chain. Cytochrome c interacts with apoptotic protease activating factor-1 (Apaf-1) and pro-caspase-9, thus forming an apoptosome, which induces programmed death of the target cell.²

It has become evident that AML cells are capable of expressing surface proteins such as programmed death-1 (PD-1) receptor ligands (PD-Ls) 1 and 2 and CD86, the ligand of cytotoxic T-cell antigen 4 (CTLA4).¹ T helpers and CTCs/NK cells express PD-1 receptors on their surfaces. AML cell surface-based PD-1 ligands 1 and 2 (PD-L(i)) interact with PD-1 on lymphoid cell surfaces. As a result, T helper cells stop producing the IL-2 required for the activation of both CTCs and NK cells. PD-1 signaling attenuates the protein kinase C-θ loop, thus preventing the activation of transcription factors nuclear factor (NF)-κB and activator protein-1, which are required for IL-2 production.⁴

However, the interaction of PD-Ls with PD-1 on the surfaces of NK cells and CTCs leads to their rapid inactivation, and, as a result, they lose capacity to kill AML cells.¹ In addition, AML cells are often capable of expressing the CTLA4 ligand CD86. The interaction of CD86 and CTLA4 rapidly leads to the inactivation of effector T cells.^{1, 5}

Thus, one could conclude that CD86 and PD-Ls 1 and 2 are involved in the formation of immunological synapses with both regulatory and cytotoxic lymphoid cells, leading to downregulation of the biochemical activation of CTCs and NK cells. The direct interaction of PD-Ls and CD86 with CTCs and NK cells leads to the loss of their anti-cancer activities. This process is shown schematically in Fig. 1.

Fig. 1.

AML cells suppress IL-2 production and the activity of cytotoxic lymphoid cells via PD-1 and CTLA4 receptors

Recent evidence has also demonstrated the capacity of AML cells to downregulate the activity of cytotoxic lymphoid cells through lymphocyte-activation gene 3 (LAG-3), a homolog of CD84. AML cells have been reported to induce the exhaustion of cytotoxic lymphoid cells through LAG-3; however, detailed mechanisms of this event remain to be elucidated.^{1, 5}

Recently, it has become evident that the immune receptor Tim-3 (T-cell immunoglobulin and mucin domain containing protein 3) is involved in protecting AML cells from host immune surveillance.^{2, 6} Tim-3 has a natural ligand, galectin-9 (a tandem protein that contains two receptor-binding domains fused together by a peptide linker), which was suggested to form an autocrine loop with the receptor.⁷ When present on the cell surface, galectin-9 induces Tim-3 downstream signaling, which includes activation of pathways responsible for cell survival.^7–9 First, this signaling includes activation of transcription factor NF-κB,⁷ translational pathways controlled by mammalian target of rapamycin (mTOR) and hypoxic signaling required for the adaptation of AML cells to stress conditions and their survival in general.^{8, 9} The Tim-3–galectin-9 complex has also been reported to activate the β-catenin pathway, which, together with NF-κB, controls AML cell self-renewal.⁷ Taken together, one may conclude that galectin-9 mediates survival signaling through Tim-3 (Fig. 2).

Fig. 2.

The Tim-3–galectin-9 pathway regulates both intracellular AML cell survival signaling and immune escape

Galectin-9 lacks a secretory domain and thus requires a trafficker before it can be taken to the cell surface and then secreted.^{2, 10} We have recently found that AML cells, but not healthy leukocytes,¹¹ express the neuronal receptor latrophilin 1 (LPHN1). LPHN1 is expressed in hematopoietic stem cells but disappears upon maturation unless the cells undergo malignant transformation into AML cells. Using its natural ligands (e.g., fibronectin leucine-rich transmembrane protein 3 (FLRT3)), LPHN1 facilitates the exocytosis of Tim-3–galectin-9, which then triggers cell survival signaling. However, Tim-3, either on its own or in complex with galectin-9, can also be proteolytically shed from the surface of AML cells, thus leading to the secretion of both proteins. Galectin-9 interacts with NK cells and CTCs (most likely though Tim-3).² This interaction leads to the impairment of the cytotoxic activity of NK cells and the killing of CTCs. Interestingly, NK cells produce interferon-γ (IFN-γ) in response to stimulation with galectin-9. IFN-γ induces the activation of IDO1 (indoleamine 2,3-dioxygenase), an enzyme that converts L-tryptophan into formyl-L-kynurenine, which is then degraded into L-kynurenine and released.¹² L-kynurenine impairs the cytotoxic activity of NK cells.²

Intriguingly, IFN-γ is also known to induce the expression of PD-Ls,¹³ which might further promote the capacity of AML cells to protect themselves against host immune surveillance.

Soluble Tim-3 released by AML cells is capable of downregulating IL-2 secretion by Th cells acting via a receptor that remains to be identified.² This downregulation prevents the activation of cytotoxic lymphoid cells. Importantly, the secretion of Tim-3 and galectin-9 allows AML cells to suppress cytotoxic lymphoid cells at a distance, thus minimizing the direct interaction with them and allowing AML cells to “focus on” self-renewal, leading to rapid disease progression. The functioning of the Tim-3–galectin-9 secretory and signaling pathway in AML cells is summarized in Fig. 2.

Importantly, stress associated with the events described above leads to release of HMGB1 (high-mobility group box 1) protein by AML cells, which ultimately triggers the production of IL-1β by healthy leukocytes.¹⁴ IL-1β has been reported to induce the expression and production of stem cell factor (SCF) by epithelial cells via the mTOR pathway and hypoxic signaling.¹⁵ SCF is a major hematopoietic growth factor that controls AML progression, thus becoming highly oncogenic.¹⁵ In this way, AML cells employ body systems to produce factors required for their proliferation/disease progression.^{14, 15}

Taking these results together, it is clear that AML cells implement comprehensive mechanisms to escape immune surveillance and facilitate disease progression. Pharmacological targeting of the biochemical pathways responsible for immune escape will enable the human immune system to potentially cure AML, with the goal of avoiding aggressive chemotherapy and bone marrow transplantation. Therefore, the design and development of new strategies for anti-AML immunotherapy are major focuses in current applied AML research. It is also vital to investigate whether other cancers utilize similar mechanisms because certain solid tumors (e.g., colon cancer¹⁶) have already been reported to use the Tim-3–galectin-9 loop for immune evasion.

Competing interests

The authors have no competing interests to declare.