Human AML activates the aryl hydrocarbon receptor pathway to impair NK cell development and function

Abstract

Acute myeloid leukemia (AML) can evade the mouse and human innate immune system by suppressing natural killer (NK) cell development and NK cell function. This is driven in part by the overexpression of microRNA (miR)-29b in the NK cells of AML patients, but how this occurs is unknown. In the current study, we demonstrate that the transcription factor aryl hydrocarbon receptor (AHR) directly regulates miR-29b expression. We show that human AML blasts activate the AHR pathway and induce miR-29b expression in NK cells, thereby impairing NK cell maturation and NK cell function, which can be reversed by treating NK cells with an AHR antagonist. Finally, we show that inhibition of constitutive AHR activation in AML blasts lowers their threshold for apoptosis and decreases their resistance to NK cell cytotoxicity. Together, these results identify the AHR pathway as a molecular mechanism by which AML impairs NK cell development and function. The results lay the groundwork in establishing AHR antagonists as potential therapeutic agents for clinical development in the treatment of AML.

Graphical abstract

Figure 1.

AHR Ligands are produced by AML cells lines and primary AML blasts. Conditioned media were harvested from AML cell lines (A), primary human AML blasts (B), or from murine leukemic cells (C) and used to treat HepG2 liver cells. RNA was isolated from HepG2 cells following 24-hour treatment, and CYP1A1 gene expression was evaluated by qPCR in each cohort as an indicator of AHR pathway activation. Data presented as expression relative to CYP1A1 expression in HepG2 cells treated with media only. A minimum of 2 independent experiments were conducted, representative figure depicted. (D) Kaplan-Meier overall survival curve comparing patients with higher mRNA expression of AHR-regulated genes (CYP1A1, CYP1A2, CYP1B1, UGT1A1, CYP2S1, and AHRR) relative to the mean expression in adult de novo AML. These data were generated through cBioPortal interface, based on data generated by The Cancer Genome Atlas Research Network. Student t test, *P < .05; **P < .01; ***P < .001. Error bars indicate standard deviation (SD).

Figure 2.

AHR regulates miR-29b. (A) Illustration depicting 2 predicted AHR-binding consensus sequences, also known as DREs, in the miR-29b promoter, highlighted in the red boxes. pGL3 luciferase reporter plasmids were cloned into HepG2 cells with seed sequence for either CYP1A1 (positive control) or miR-29b (B), miR-29a/b1 or miR-29b2/c (C), or miR-29b with the 5′ AHR site or the 3′ AHR binding site within the miR-29b promoter mutated (D). Each group was then treated with DMSO or FICZ (an AHR agonist). (E) pGL3 luciferase reporter plasmids were cloned into the miR-29b promoter in HepG2 cells, and AHR was knocked down in the same cells. The scrambled control (siCTRL) and the siAHR were treated with DMSO or FICZ and luciferase was measured. (F) CD56⁺/Lineage negative NK cells were isolated from PB and treated with vehicle DMSO vehicle control or FICZ. ChIP was performed with anti-AHR or isotype control antibody. The anti-AHR ChIP product was evaluated for miR-29b promoter expression in by qPCR to validate association between AHR and the miR-29b promoter. Data reported as expression relative to control. (G) Primary PB allogeneic NK cells were cultured with either media only or AML conditioned media, and miR-29b ChIP was performed similar to panel F. Minimum of 2 independent studies performed with representative figures depicted. *P < .05; ***P < .0001. Error bars indicate SD.

Figure 3.

miR-29b is expressed by NKDIs and regulates NK cell development. (A) Freshly isolated tonsillar stage 3 cells were incubated for 24 hours with FICZ or DMSO, and miR-29b expression was determined by quantitative RT-PCR. (B) Stage 3 NKDI cells were cultured ex vivo in the presence of IL-15 and IL-1β and infected with an empty lentiviral vector (empty vector; negative control), cultured with the AHR inhibitor CH223191 (positive control), or infected with the miR-29b lentiviral knockdown (miR-29B KD). In total, 6 independent studies were performed with stage 3 NK cells from 10 primary human donors. (C) A representative figure is depicted. Student t test, *P < .05. Error bars indicate SD.

Figure 4.

NK cell development is altered by AML cells. (A) Graphical illustration of NK cell development coculture in vitro. (B) Postculture flow cytometry analysis of CD94⁺ mature NK cells in the presence of AML vs control. (C) Stage 3 NK cells were cocultured via transwell with MV411 AML cells with and without treatment with the AHR inhibitor, CH223191 (CH); n = 14 independent donors. Stage 3 (S3) Lin⁻CD34⁻CD117⁺CD94⁻NKp80⁻. (D) Post-coculture, IFN-γ was evaluated following phorbol 12-myristate 13-acetate/ionomycin stimulation; n = 5 independent donors. Student t test, *P < .05; **P < .01; ***P < .001; ****P < .0001. Error bars indicate SD.

Figure 5.

Effect of AHR ligands on miR-29b expression in NK cells. (A-B) CD56⁺/Lineage negative NK cells were treated with DMSO (vehicle control) or FICZ (AHR agonist), or conditioned media obtained from U937 AML cells for 4 hours, and miR-29b expression was evaluated by RT-PCR. (C) Primary NK cells were cultured alone or in transwell with AML blasts either untreated or treated with CH223191. miR-29b expression was evaluated by qPCR, representative donor depicted, n = 3. (D) NK cells were treated with DMSO (vehicle control), AHR agonist (FICZ), or AHR antagonist (CH223191) for 48 hours, stimulated with IL-12 and IL-18, and evaluated for the production of soluble IFN-γ by enzyme-linked immunosorbent assay (Student t test, *P > .05). Error bars indicate SD.

Figure 6.

Effect of AHR modulation on AML blasts and NK cell function. (A) AHR expression profiles of AML cell lines and primary AML samples were evaluated by flow cytometry. (B-C) Primary human AML samples were either treated with DMSO (vehicle control) or CH223191 for 48 hours, and cell survival by trypan blue counting and apoptosis by annexin V/propidium iodide (PI) flow cytometric evaluation, representative donor, n = 5 primary donors and 4 AML cell lines (*P = .03).

Figure 7.

Blocking AHR on AML cells increases susceptibility to NK cell IFN-y production and cytotoxicity. (A) EOL-1 cells were treated for 5 days with AHR agonist (FICZ) or AHR antagonist (CH223191) and then washed and cocultured with NK cells in the presence of IL-12 and IL-18 (10 ng/mL) for 48 hours and evaluated for the production of soluble IFN-γ by enzyme-linked immunosorbent assay. (B) Primary AML blasts were cultured for 5 days with AHR antagonist (red, CH223191) or vehicle control (blue, DMSO). Cells were then washed to remove antagonist. Normal donor NK cells were cocultured with the primary AML targets for 4 hours in a standard chromium release assay. Six representative primary AML donors depicted (n = 10 total evaluated). Student t test, *P < .05; **P < .01; ***P < .001. Error bars indicate SD.