Effect of decitabine on expression of PD-L2 gene in AML1-ETO positive acute myeloid leukemia cells

  Background   Refractory and relapsed acute myeloid leukemia patients carrying AML1-ETO fusion gene have a poor prognosis. Programmed cell death-ligand 2 (PD-L2) is an important molecule involved in leukemia immune escape. Studying the effect of demethylation agents on immune checkpoint molecules in the leukemia cells will help to optimize the treatment regimen.

  Objective   To analyze the DNA methylation level of PD-L2 gene promoter in AML1-ETO-positive AML cells and investigate the effect of DNA methyltransferase inhibitor decitabine (DAC) on the expression of PD-L2.

  Methods   The AML1-ETO-positive AML cell lines Kasumi-1 and two pairs of AML1-ETO manipulated cell lines (AML1-ETO silenced: SKNO-1-PGK, SKNO-1-siA/E and AML1-ETO induced: U937-MT, U937A/E, U937A/E + ZnSO₄) were used as the model. The cells were treated with DAC with a final concentration of 2.5 μmol/L or 0.1% dimethyl sulfoxideas (vehicle control) for 72h. Kasumi-1 and SKNO-1-PGK cells were treated with different concentrations of DAC (0.25 μmol/L, 1.0 μmol/L and 2.5 μmol/L). DNA methylation levels of the PD-L2 gene promoter in Kasumi-1 and SKNO-1-PGK cell lines were detected by bisulfite sequencing method. Reverse transcription and quantitative PCR technology was used to detect PD-L2 mRNA levels in each cell lines.

  Results   The PD-L2 promoter region in Kasumi-1 and SKNO-1-PGK cell lines showed high DNA methylation levels (94.3% and 88.6%, respectively) and the promoter methylation in PD-L2 decreased upon DAC treatment (90.7% and 83.6%, respectively). The relative mRNA expression levels of PD-L2 mRNA in Kasumi-1, SKNO-1-PGK, SKNO-1-siA/E and U937A/E cell lines treated with 2.5 μmol/L of DAC were significantly higher than those in control group (all P<0.05). Especially, PD-L2 expression increased more in cell lines silencing the AML1-ETO gene (SKNO-1-siA/E was 35.80% more compared to SKNO-1-PGK, P=0.031) and less in cell lines overexpressing the AML1-ETO gene (U937A/E + ZnSO₄ decreased 95.51% compared to U937A/E, P=0.036) after DAC treatment. PD-L2 mRNA expression increased in a concentration-dependent manner upon DAC treatment in Kasumi-1 and SKNO-1-PGK cell lines (Kasumi-1: r²=0.87; SKNO-1-PGK: r²=0.93, all P<0.05).

  Conclusion   The expression of PD-L2 in AML1-ETO-positive AML cells is regulated by DNA methylation and AML1-ETO might be involved in the inhibition of PD-L2 gene expression.