Aberrant EPHB4 gene methylation and childhood acute lymphoblastic leukemia

Abstract

The present study aimed to investigate the association between aberrant DNA methylation of the promoter region of the ephrin type-B receptor 4 (EPHB4) gene and the development of childhood acute lymphoblastic leukemia (ALL). Bisulfite sequencing polymerase chain reaction (BSP) was performed to determine the methylation density of cytosine-guanine pair islands in the promoter region of EPHB4, in bone marrow samples from 40 children with ALL. The mRNA and protein expression levels of EPHB4 were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. A total of 10 children with idiopathic thrombocytopenic purpura (ITP) were recruited as controls. The results revealed that the average methylation density of the bone marrow samples from the patients with ALL was significantly higher, compared with the patients with ITP (P=0.046). The relative mRNA expression levels of EPHB4 in the patients with ITP (25.08±4.03) and the patients with ALL without methylation (12.33±2.16) were significantly higher, compared with that observed in the patients with ALL with methylation (6.48±2.73; P<0.01). Pearson analysis revealed a significant negative linear correlation between EPHB4 gene methylation and its expression levels (r=−0.957; P<0.01). Western blot analysis indicated that EPHB4 protein expression levels were low in the methylated ALL samples. An evaluation of the two-year disease-free survival (DFS) of the patients with ALL was performed, which revealed that the patients with unmethylated ALL exhibited a significantly higher two-year DFS rate, as compared with patients with methylated ALL (P=0.036). These results suggest that the methylation of the EPHB4 gene is prevalent in childhood ALL and may result in expressional inactivation, which consequently promotes ALL pathogenesis and is associated with an unfavorable prognosis. Therefore, the EPHB4 gene may function as a potential tumor suppressor in childhood ALL.

The Clinical Relevance of the EPH/Ephrin Signaling Pathway in Pediatric Solid and Hematologic Malignancies

“…Specifically, the observation that a high expression of EPHA2 in neuroblastoma [103], EPHB1 in glioma [79], EPHB6, ephrin-B2, and ephrin-B3 in neuroblastoma [101] correlates with a better prognosis is in line with a tumor-suppressive role of these EPHs. EFNB1 has been identified among downregulated DEGs in children diagnosed with AML [134], while bone marrow samples from pediatric AML patients are characterized by EPHB1 promoter hypermethylation with subsequent decreased EPHB1 expression [136], while analogously for the case of pediatric ALL, the observed increased EPHB4 methylation resulting in decreased EPHB4 expression may contribute to leukemia development and progression [131]. Interestingly, as it is observed for the case of EPHB4, the same EPH can exert either a pro-tumorigenic (i.e., in neuroblastoma and rhabdomyosarcoma [106,120]) or anti-tumorigenic function (i.e., in ALL [131]) depending on the type of neoplasm it is expressed.…”

“…EFNB1 has been identified among downregulated DEGs in children diagnosed with AML [134], while bone marrow samples from pediatric AML patients are characterized by EPHB1 promoter hypermethylation with subsequent decreased EPHB1 expression [136], while analogously for the case of pediatric ALL, the observed increased EPHB4 methylation resulting in decreased EPHB4 expression may contribute to leukemia development and progression [131]. Interestingly, as it is observed for the case of EPHB4, the same EPH can exert either a pro-tumorigenic (i.e., in neuroblastoma and rhabdomyosarcoma [106,120]) or anti-tumorigenic function (i.e., in ALL [131]) depending on the type of neoplasm it is expressed. We should note, though, that for certain EPH/ephrin members, such as EPHB1 in medulloblastoma [69] and EPHB2 in ependymoma [95], the reported variable levels of expression render the characterization of their role in tumorigenesis ambiguous.…”

“…In a study by Li et al, including 40 pediatric patients with ALL and 10 controls, protein expression analysis in bone marrow samples revealed a higher frequency of EPHB4 methylation in ALL patients. This methylation profile negatively correlated with patients' prognoses, therefore highlighting a tumor-suppressive effect of EPHB4 [131]. Another study by El Zawily et al, comparing 117 pediatric T-ALL samples with the controls, reported an elevated EPHB6 expression in T-ALL patients.…”

EPH/Ephrin Signaling in Normal Hematopoiesis and Hematologic Malignancies: Deciphering Their Intricate Role and Unraveling Possible New Therapeutic Targets

“…An association with an unfavorable prognosis has also been observed. Therefore, the authors proposed that the EPHB4 gene may function as a potential tumor suppressor in childhood ALL [147]. Another study by Kuang et al identified hypermethylation of multiple EPH and ephrin genes (i.e., EPHA2, -A4, -A5, -A6, -A7, -A10, EPHB1, -B2, -B3, -B4, EFNA1, -A3, -A5, and EFNB1 and -B2) in leukemia cell lines and primary ALL BM samples.…”

GSTT1 Null Genotype and Susceptibility to Children Acute Leukemia in Chinese Population: Evidence from a Meta-Analysis

“…Sixty-five records were identified about the relationship of GSTT1 polymorphisms and children acute leukemia risk. According to the selection criteria, 7 studies [10][11][12][13][14][15][16] were suitable and the remaining was removed. The publication year of all included articles ranged from 2003 to 2013.…”