DNA hypermethylation resulting in aberrant epigenetic silencing plays an important role in the oncogenesis of many cancer types, including acute myelogenous leukemia (AML).4 The modulation of NK cell receptors and their cognate ligands is a known mechanism of immune escape in AML, and some membrane proteins, such as killer immunoglobulin-like receptors (KIR), are known to be transcriptionally regulated by DNA methylation of their promoter regions. Thus, restoring proper expression of immunoreceptors or their ligands with immunosensitizing drugs is an attractive approach to improving cancer immunotherapy. The cytidine analog 5-aza-2′-deoxycytidine (decitabine, DAC) has both a hypomethylating effect at low doses when incorporated into DNA and a cytotoxic effect at higher doses as a result of interfering with translation when incorporated into RNA. Thus, decitabine has been used at higher doses for its direct anti-leukemic effect, and is being tested at low doses for its ability to correct the malignant gene expression phenotype. A known benefit of hypomethylating agents is their ability to sensitize AML blasts to lysis by NK cells. However, there is little information on the direct effect of hypomethylating agents on NK cell phenotype, proliferation, survival, or function. We recently described a method for inducing robust proliferation of NK cells, enabling us to study the hypomethylating effects of decitabine.
To distinguish direct toxicity of the decitabine from its hypomethylating effect, and promote hypomethylation during proliferation, decitabine was added to human peripheral blood NK cells at concentrations from 0.02 to 5 μM under either static or proliferation-inducing culture conditions. After 5 days, NK cells were assessed for viability, proliferation, cytotoxicity, expression of major activating and inhibitory receptors, and global DNA methylation.
Increasing concentrations of decitabine not only causes increased expression of KIR and the activating receptor NKp44, but also causes decreased viability, proliferation, and expression of the activating receptor NKG2D. Decitabine treatment results in a biphasic effect in overall NK cell lytic function, which correlates with a biphasic pattern of global hypomethylation.
Decitabine affects the expression of activating and inhibitory receptors in NK cells at low concentrations when exposed during cell proliferation. High doses of decitabine decrease NK cell proliferation and viability, likely through direct inhibition of mRNA transcription. The results of these combined effects leads to a biphasic response in hypomethylation and cytotoxicity. This suggests that optimal immunomodulation with decitabine occurs at low dose ranges and that high doses abrogate this effect through inhibition of proliferation and direct toxicity to NK cells.