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. Disclosure of potential conflicts of interest is found at the end of this article.