Rapamycin downregulates NKG2D ligands in acute myeloid leukemia cells via an activation of the STAT3 pathway: a potential mechanism for rapamycin-induced immune escape in leukemia

Abstract

Background: The constitutive activation of the mammalian target of rapamycin (mTOR) is involved in the pathogenesis of many cancers. Rapamycin (RAPA), a specific inhibitor of mTOR, has been applied to the clinical treatment of tumors, and its anti-leukemia effect has also been confirmed.

Methods: We detected apoptosis and the NKG2D ligands expression in acute myeloid leukemia (AML) cells using flow cytometry and investigated the cytotoxicity of AML cells that had been co-cultured with natural killer (NK) cells using CFSE staining. We evaluated the signal pathways with a western blot assay.

Results: In this study, we found that RAPA can significantly inhibit the proliferation of AML cells. Further studies showed that the use of RAPA alone reduced the expression of NKG2D ligands on the membranes of HL-60 and THP-1 AML cells. Also, RAPA blocked the upregulation of the NKG2D ligand when AML cells were cultured with the demethylation drug decitabine (DAC). We found that RAPA decreased the expression of the NKG2D ligands by inducing the STAT3 phosphorylation of AML cells.

Conclusions: The discovery of this mechanism might further optimize the clinical use of RAPA for the treatment of AML.

Keywords: NKG2D ligand; Rapamycin (RAPA); STAT3; acute myeloid leukemia (AML); immune escape.

Figure 1

Anti-leukemia effect of RAPA on AML cells. (A) Proliferation inhibition efficiency of various concentrations of RAPA (0, 0.01, 0.1, 1.0 µg/mL) on AML cells (HL-60 and THP-1) for 24, 48, and 72 h. AML cells were incubated in 96-well plates at a density of 1×10⁴ cells per well for 24, 48, and 72 h, and then CCK-8 solution was added for a further 4 h incubation. Results are reported as mean ± SD of the 3 independent experiments. Statistical significance was reported as **, P<0.01, ***, P<0.001; (B) HL-60 and THP-1 cells treated with different doses of RAPA (0, 0.01, 0.1, 1.0 µg/mL) were analyzed by FACS for the percentage of apoptotic cells for 48 h. Q1, dead cells; Q2, advanced apoptosis cells; Q3, living cells; Q4, early apoptosis cells; (C) cell apoptosis of AML cells (HL-60 and THP-1) following RAPA treatment. Each column and error bar represent the mean ± SD of apoptosis level (n≥2). RAPA, rapamycin; AML, acute myeloid leukemia; SD, standard deviation.

Figure 2

RAPA downregulates the cytotoxic sensitivity of AML to NK cells by decreasing NKG2DL expression on AML cells. (A) Flow cytometry results of the expression of 5 NKG2D ligands expressed on both AML cells (HL-60 and THP-1) after various concentrations of RAPA (0, 0.01, 0.1, 1.0 µg/mL) treatment for 24 h. Black line, IgG; grey line, Con; red line, 0.01 µg/mL; green line, 0.1 µg/mL; blue line, 1.0 µg/mL; (B,C) the downregulation of rMFI levels of 5 NKG2D ligands (MICA/B, ULBP1-4) in HL-60 cells and THP-1 cells after RAPA treatment; (D) flow cytometry of cytotoxic sensitivity of HL-60 cells to NK cells. After RAPA treatment for 24 h, the cytotoxic sensitivity of HL-60 cells to NK cells significantly declined; (E) RAPA downregulated the cytotoxic sensitivity of HL-60 cells to NK cells at different E:T ratios. HL-60 cells were previously incubated with 0.1 µg/mL RAPA for 24 h and then co-cultured at a different effector-target ratio (E:T ratio). Results are reported as mean ± SD of the 3 independent experiments (B,C,E). *, P<0.05 vs. control group; **, P<0.01 vs. control group. RAPA, rapamycin; AML, acute myeloid leukemia; NK, natural killer; IgG, immunoglobulin G; rMFI, relative mean fluorescence intensity; SD, standard deviation.

Figure 3

RAPA inhibited the expression of NKG2D ligand on the STAT3 signaling pathway. (A,B) RAPA upregulated the activity of STAT3 and ERK in HL-60 and THP-1 cells. AML cells were exposed to 0.1 µg/mL RAPA for 24 h. Cell lysates were prepared and subjected to western blot assay for p-STAT3 (Tyr705 and Ser727), total STAT3 protein, p-ERK, and total ERK. The phosphorylated STAT3 and ERK in HL-60 cells (A) and THP-1 cells (B) were increased after the treatment of RAPA; (C) without changing the basic expression of NKG2D ligand, STAT3 specific inhibitor upregulated the expression of ULBP3. HL-60 cells were treated with 0.1 µg/mL RAPA, 0.25 µmol/L STAT3 VII inhibitor or 0.1 µg/mL RAPA +0.25 µmol/L STAT3 VII inhibitor for 24 h. The expression of NKG2D ligands was analyzed using flow cytometry. Results are reported as mean ± SD of the 3 independent experiments. *, P<0.05 vs. RAPA group. RAPA, rapamycin; AML, acute myeloid leukemia; SD, standard deviation.

Figure 4

RAPA antagonized the anti-leukemia effect of DAC by upregulating phosphorylation of STAT3 protein. (A) Flow cytometry results of the expression of NKG2D ligand expressed on HL-60 cells after treatment with DAC or cotreatment with DAC and RAPA for 48 h. Red, IgG; blue, Con; orange, 0.2 µmol/L DAC; green, 0.1 µg/mL RAPA +0.2 µmol/L DAC; (B) the downregulation of rMFI levels of 5 NKG2D ligands (MICA/B, ULBP1-3) in HL-60 cells after treated with DAC or DAC combined with RAPA. After HL-60 cells were treated with a different concentration of DAC+0.1 µg/mL RAPA, the expression of NKG2D ligands on HL-60 cells were downregulated. Even at a high concentration of DAC, it cannot further increase the NKG2D ligands expression; (C) RAPA upregulated the phosphorylated STAT3 in DAC-treated HL-60 cells. Results are reported as mean ± SD of the 3 independent experiments. *, P<0.05 vs. DAC group; **, P<0.01 vs. DAC group. RAPA, rapamycin; DAC, demethylation drug decitabine; rMFI, relative mean fluorescence intensity; SD, standard deviation.