Imprint of 5-azacytidine on the natural killer cell repertoire during systemic treatment for high-risk myelodysplastic syndrome

Abstract

5-azacytidine (5-aza) is a hypomethylating agent approved for the treatment of high-risk myelodysplastic syndrome (MDS). It is assumed to act by demethylating tumor suppressor genes and via direct cytotoxic effects on malignant cells. In vitro treatment with hypomethylating agents has profound effects on the expression of killer-cell immunoglobulin-like (KIR) receptors on natural killer (NK) cells, as these receptors are epigenetically regulated via methylation of the promoters. Here we investigated the influence of 5-aza on the NK-cell repertoire during cytokine-induced proliferation in vitro and homeostatic proliferation in vivo in patients with high-risk MDS. In vitro treatment of NK cells from both healthy donors and MDS patients with low doses of 5-aza led to a significant increase in expression of multiple KIRs, but only in cells that had undergone several rounds of cell division. Proliferating 5-aza exposed NK cells exhibited increased IFN-γ production and degranulation towards tumor target cells. MDS patients had lower proportions of educated KIR-expressing NK cells than healthy controls but after systemic treatment with 5-aza, an increased proportion of Ki-67+ NK cells expressed multiple KIRs suggesting uptake of 5-aza in cycling cells in vivo. Hence, these results suggest that systemic treatment with 5-aza may shape the NK cell repertoire, in particular during homeostatic proliferation, thereby boosting NK cell-mediated recognition of malignant cells.

Immunological effects of hypomethylating agents

“…MICA/B stress ligands) for which NK cells have activating receptors (e.g. NKG2D) [30,31]. NK cell recognition and activation against malignant cells is induced upon two conditions: the lack of recognition of MHC class I molecules by KIRs and the recognition of stress ligands by their respective activating receptors [27,30].…”

“…NKG2D) [30,31]. NK cell recognition and activation against malignant cells is induced upon two conditions: the lack of recognition of MHC class I molecules by KIRs and the recognition of stress ligands by their respective activating receptors [27,30]. It is a combination of these mechanisms that enable the downstream effects of NK cell recognition of, and cytotoxicity against, malignant or infected cells (i.e.…”

“…Promotor methylation of KIR genes has been shown to consistently suppress KIR expression on NK cells, whereas hypomethylation enables KIR expression [29,30,32]. Bisulfite sequencing of a region around the translation initiation site of the NKG2D gene in cells expressing various levels of the protein product revealed a significant correlation between methylation at this site and protein expression[31].…”

“…Sohlberg et al . also demonstrated that low-dose in vitro administration of azacitidine upregulates KIR expression on NK cells from both healthy donors and MDS patients [30]. However, in contrast to the other studies, Sohlberg et al .…”

“…However, in contrast to the other studies, Sohlberg et al . observed an amplification of NK cell-mediated cytotoxicity against K562 cells, defined by increased INF-γ production, degranulation, and killing frequency [30]. These results pertained only to Ki-67+ NK cells, an indicator of NK cells that have undergone recent activation or proliferation [30].…”