Azacytidine prevents experimental xenogeneic graft-versus-host disease without abrogating graft-versus-leukemia effects

ABSTRACT

The demethylating agent 5-azacytidine (AZA) has proven its efficacy in the treatment of myelodysplastic syndrome and acute myeloid leukemia. In addition, AZA can demethylate FOXP3 intron 1 (FOXP3i1) leading to the generation of regulatory T cells (Treg). Here, we investigated the impact of AZA on xenogeneic graft-vs.-host disease (xGVHD) and graft-vs.-leukemia effects in a humanized murine model of transplantation (human PBMCs-infused NSG mice), and described the impact of the drug on human T cells in vivo. We observed that AZA improved both survival and xGVHD scores. Further, AZA significantly decreased human T-cell proliferation as well as IFNγ and TNF-α serum levels, and reduced the expression of GRANZYME B and PERFORIN 1 by cytotoxic T cells. In addition, AZA significantly increased Treg frequency through hypomethylation of FOXP3i1 as well as increased Treg proliferation. The latter was subsequent to higher STAT5 signaling in Treg from AZA-treated mice, which resulted from higher IL-2 secretion by conventional T cells from AZA-treated mice itself secondary to demethylation of the IL-2 gene promoter by AZA. Importantly, Tregs harvested from AZA-treated mice were suppressive and stable over time since they persisted at high frequency in secondary transplant experiments. Finally, graft-vs.-leukemia effects (assessed by growth inhibition of THP-1 cells, transfected to express the luciferase gene) were not abrogated by AZA. In summary, our data demonstrate that AZA prevents xGVHD without abrogating graft-vs.-leukemia effects. These findings could serve as basis for further studies of GVHD prevention by AZA in acute myeloid leukemia patients offered an allogeneic transplantation.

KEYWORDS:

• Azacytidine
• graft-vs.-host disease
• graft-vs.-leukemia effect
• NOD-scid IL-2Rγ^null
• regulatory T cells

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are very grateful to Sandra Ormenese and Raafat Stephan from the Imaging and Flow Cytometry Platform of the GIGA for help with flow cytometry analyses, to the Genomics platform of the GIGA for sequencing and to Emmanuel Di Valentin from the Viral Vectors Platform of the GIGA for help with THP-1 cells transfections. The authors are also grateful to Coline Daulne, for excellent technical assistance. GE, GF and LD are Télévie Research Assistants, and SL and FB are senior research associates of the National Fund for Scientific Research (FNRS) Belgium. SHB is funded by the Fonds Wetenschappelijk Onderzoek (FWO).

Funding

This work was supported by the National Fund for Scientific Research (FNRS) under grant T.0069.15; the Leon Fredericq fund and Anti-Cancer Center at the University of Liège.

Author contributions

Study design: FBa, GE, SHB; Data analyses: GE; Mouse experiments: GE, GF, MH, LD, SD, PD; Flow cytometry design/analyses: GE, SHB; Cell culture, RT-qPCR and IL2 promoter methylation sequencing: GE, LD, GF. FOXP3i1 sequences methylation: SDH, SL; Histology: LdL; Ms writing: GE, FBa, and SHB; Manuscript editing and approval of the manuscript: all authors.