Hypermethylation of CXCR4 Promoter in CD34+ Cells from Patients with Primary Myelofibrosis

Abstract

Constitutive mobilization of CD34+ cells in patients with primary myelofibrosis (PMF) has been attributed to proteolytic disruption of the CXCR4/SDF-1 axis and reduced CXCR4 expression. We document here that the number of circulating CD34+/CXCR4+ cells in PMF patients, as well as the cellular CXCR4 expression, was directly related to CXCR4 mRNA level and that reduced CXCR4 mRNA level was not due to SDF-1-induced downregulation. To address whether epigenetic regulation contributes to defective CXCR4 expression, we studied the methylation status of the CXCR4 promoter using methylation-specific polymerase chain reaction and methylation-specific sequencing in the JAK2V617F-positive HEL cell line and in CD34+ cells. We found that CD34+ cells from PMF patients, unlike those from normal subjects, presented hypermethylation of CXCR4 promoter CpG island 1. Following incubation with the demethylating agent 5-Aza-2′-deoxycytidine (5-AzaD), the percentage of PMF CD34+ cells expressing CXCR4 increased 3–10 times, whereas CXCR4 mRNA level increased approximately 4 times. 5-AzaD-treated PMF CD34+ cells displayed almost complete reversal of CpG1 island 1 hypermethylation and showed enhanced migration in vitro in response to SDF-1. These data point to abnormal methylation of the CXCR4 promoter as a mechanism contributing to constitutive migration of CD34+ cells in PMF.Disclosure of potential conflicts of interest is found at the end of this article.