Differentiation-promoting drugs up-regulate NKG2D ligand expression and enhance the susceptibility of acute myeloid leukemia cells to natural killer cell-mediated lysis
Introduction
Epigenetic alterations leading to transcriptional silencing of regulators of cell growth and differentiation have been implicated in the pathogenesis of acute leukemias [1]. In contrast to oncogenic changes produced by gene fusions and mutations, epigenetic changes are thought to be reversible and, therefore, amenable to pharmacologic intervention with anti-neoplastic agents counteracting DNA modifications and chromatin remodelling [2], [3]. All-trans-retinoic acid (ATRA), which causes disassembly of transcriptional co-repressor complexes with histone deacetylase (HDAC) activity, is very effective in the treatment of acute promyelocytic leukemia, inducing terminal granulocytic differentiation and complete disease remission [4]. The hypomethylating agent 5-aza-2′-deoxycytidine (Aza), the HDAC inhibitor trichostatin A (TSA), vitamin D3 (VD3) binding to specific nuclear receptors, and bryostatin-1 (Bryo-1) acting through protein kinase C-mediated signaling [5], [6], [7], [8], all promote hematopoietic differentiation, as documented with cell lines and primary leukemic cells in vitro[9], [10], [11], [12], [13]. In clinical trials, hematologic responses to monotherapies and drug combination regimens have been reported, although their efficacy has so far been limited [14], [15], [16], [17]. As large numbers of genes are affected by these drugs [9], [18], [19], defining target molecules relevant for therapeutic effects has been difficult.
NKG2D is a well characterized activating immunoreceptor expressed by all NK cells and T cell subsets [20], [21]. Human NKG2D recognizes several cell surface ligands belonging to major histocompatibility complex class I chain-related (MIC) and UL-16-binding protein (ULBP) families [22], [23]. Engagement of NKG2D by these ligands induces secretion of proinflammatory cytokines and triggers the NK cell cytolytic program against tumor targets [23]. In human tumors of epithelial origin, the stress-inducible MIC ligands are up-regulated, marking the tumor tissue for destruction by NK cells [24]. In contrast to findings with epithelial tumors, malignant blood cells in acute myeloid leukemia (AML) express only low levels of NKG2D ligands and evade NK-mediated immunosurveillance [25], [26], [27]. Our studies demonstrated that inadequate ligand expression, which compromises leukemia recognition by NKG2D receptor-carrying cells, is a consequence of hematopoietic maturation arrest associated with malignant transformation [26]. The possibility to influence NKG2D ligand expression and NK-mediated antileukemic responses by pharmacological induction of cell differentiation in AML has not previously been explored.
In this work, we provide evidence that agents promoting the myelomonocytic differentiation of the human AML cell line HL60 and primary AML blasts increase the expression of ULBP ligands, primarily ULBP1, rendering leukemic cells more sensitive to recognition and killing by cytotoxic NK cells. These data indicate that NKG2D ligands represent relevant targets of drugs which can modify epigenetic transcriptional mechanisms in leukemia. Clinical evaluation of immunomodulatory properties of these drugs may be particularly useful when combined with NK-based immunotherapy against AML.
Section snippets
Differentiating compounds
The following drugs were used: 5-aza-2′-deoxycytidine (Aza) at 1 μM (ICN Biomedicals GmbH, Eschwege, Germany), trichostatin A (TSA) at 60 ng/mL (Sigma, Saint Louis, USA), 1-alpha,25-dihydroxy-vitamin D3 (VD3) at 0.1 μM (Merck Biosciences Ltd., Beeston, UK), bryostatin-1 (Bryo-1) at 10 nM (LKT Laboratories, St. Paul, USA), all-trans-retinoic acid (ATRA) at 1 μM (Sigma).
AML patients
Peripheral blood (PB) was obtained with informed consent from patients with AML at diagnosis in compliance with the guidelines of the
Differentiating drugs up-regulate cell surface expression of ULBP in HL60 cells
Since incomplete hematopoietic maturation of AML blasts is associated with low cell surface density of NKG2D ligands [26], we investigated whether ligand expression can be up-regulated by pharmacological compounds with a cell-differentiating function. In search for the most effective stimulatory conditions, the human AML cell line HL60 was exposed to Aza, TSA, VD3, Bryo-1, and ATRA, and combinations thereof comprising drugs which act through distinct mechanisms of transcriptional de-repression,
Discussion
Although progress has been made in the management of acute leukemias, most patients who fail to respond to front-line therapies with cytostatic agents and stem cell transplantation, or who relapse after an initial response, die from progressive disease [29], [30]. Novel treatment approaches exploit adoptive immunotherapy with donor-derived NK cells to generate alloreactive graft-versus-leukemia responses and eliminate the residual malignant clones in transplanted patients [31]. However, a very
Acknowledgements
We thank AMGEN and Novartis for recombinant human growth factors, D. Cosman for mAbs, A. Gratwohl for providing clinical AML samples, and J. Schwaller for critically reading the manuscript. This work was supported by grants from the Krebsliga beider Basel (7/2003), Swiss National Science Foundation (4046-058689 and 3100-067072), Swiss Cancer League (OCS-01664-02-2005) and Stiftung zur Krebsbekämpfung No. 216.
Contributions. Rohner A, Langenkamp U, and Siegler U contributed to design, generated,
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