Valproic acid exerts differential effects on CXCR4 expression in leukemic cells

Abstract

We recently reported that the histone deacetylase inhibitor, valproic acid (VPA), increases CXCR4 receptor expression and function in cord blood hematopoietic stem/progenitor cells (HSPC) and the immature, highly CD34-positive AML cell lines KG-1a and KG-1. In this study, we investigated whether VPA influences CXCR4 in CD34-negative AML cell lines (promyelocytic HL-60 and monocytic THP-1), as well as both CD34-positive and CD34-negative primary AML cells. We found that VPA (i) diminishes CXCR4 expression and chemotaxis in HL-60 cells and in the CD34-negative subtypes of primary AML cells and (ii) increases CXCR4 expression and function in the highly CD34-positive subtypes of primary AML cells. Hence, we suggest that VPA exerts different effects on CXCR4 depending on cell maturation status, and this novel finding may have important implications for AML therapy.

Introduction

The G protein-coupled chemokine receptor CXCR4 is expressed on primitive hematopoietic stem/progenitor progenitor cells (HSPC), and its ligand SDF-1α (CXCL12) is produced by cells, including stromal cells and osteoblasts, within the bone marrow (BM) microenvironment [1]. Interaction between SDF-1α and CXCR4 is known to play an essential role in mobilization, homing, retention and survival of HSPC [1], [2], [3]. HSPC expressing CXCR4 are attracted to the BM microenvironment by SDF-1α, and disruption of the SDF-1α/CXCR4 axis facilitates their mobilization to the peripheral blood. SDF-1α has been also reported to activate adhesion receptors such as CD44, very late antigen-4 (VLA-4) and lymphocyte function-associated antigen-1 (LFA-1) on HSPC, which may contribute to the homing process [4]. Thus, SDF-1α/CXCR4-mediated migration of HSPC is believed to be one of the major mechanisms for homing and, aside from its importance in this respect, this axis has also been reported to play crucial roles in the migration, abnormal proliferation and anchorage of human acute myelogenous leukemic (AML) cells in the BM [5].

AML, a heterogeneous group of diseases characterised by uncontrolled clonal proliferation of myeloid blasts with reduced capacity to differentiate into mature cells, has been reported to express CXCR4 differently in its various subtypes: lower expression level in the case of undifferentiated (M0), myeloid (M1/M2), and erythroid (M6) AML, and higher expression in more differentiated myelomonocytic (M4/M5) and promyelocytic (APL; M3) AML [6]. The expression level of CXCR4 on AML blasts was postulated to be a major prognostic factor for disease progression [7], [8]. Enhanced expression promotes cell retention, survival and growth within the BM microenvironment resulting in resistance to conventional chemotherapy and subsequent relapses [9], [10]. This indicates that modulation of CXCR4 gene expression could be clinically relevant not only in the trafficking of leukemic cells but also in their response to chemotherapeutic agents.

Gene expression can be regulated by chromatin remodelling [11]. Two enzyme classes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) regulate chromatin structure in normal cells [12] and alterations in these molecules can lead to aberrant transcriptional networks in AML. For example, abnormal recruitment of HDACs by the PML/RARa and AML1/ETO fusion proteins in APL and AML-M2, respectively, results in repression of genes relevant for differentiation by reducing the core histone acetylation level [13]. Therefore, differentiation or transcription therapy using histone deacetylase inhibitors (HDI) through chromatin modelling is under investigation as a new approach to treat hematological malignancies [14], [15], [16].

A specific and potent short-chain fatty acid HDI, valproic acid (VPA), has gained considerable attention among other HDI due to its safe and well-tolerated nature [17]. Recently, it was demonstrated that VPA induces differentiation of PML/RARa-transformed HSPC and primary AML blasts in vitro regardless of the primary genetic alteration [18]. Moreover, VPA has been reported to increase the cytotoxicity or favorable response of AML blasts in recent clinical trials [19], [20], [21], [22], [23], [24]. On the other hand, we and others have shown that VPA can exert opposite effects on HSPC, including the enhancement of proliferation and self-renewal [25], [26]. Furthermore, we found that VPA increases CXCR4 expression and chemotaxis of cord blood primitive HSPC towards SDF-1α and we suggested its use to improve homing and engraftment [27]. Moreover, a similar effect was observed in the CD34-positive human AML cell lines KG-1a and KG-1 [27] and this might explain both of the favorable and the adverse effects observed after treatment of AML with HDI. Here we have extended our investigation and examined the effect of VPA on CXCR4 (at the protein, transcriptional and functional levels) in CD34-negative promyelocytic HL-60 and monocytic THP-1 AML cell lines as well as CD34-positive and CD34-negative leukemic cells obtained from patients diagnosed with AML.