Hypermethylation of CXCR4 promoter in CD34+ cells from patients with primary myelofibrosis

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.