DOK6 promoter methylation serves as a potential biomarker affecting prognosis in de novo acute myeloid leukemia

Abstract

BackgroundDownstream of tyrosine kinase 6 (DOK6), which is specifically expressed in the nervous system, was previously recognized as an adapter only in neurite outgrowth. Recent studies also demonstrated the potential role of DOK6 in solid tumors such as gastric cancer and breast cancer. However, previous studies of DOK6 have not dealt with its roles in myeloid malignancies. Herein, we verified the promoter methylation status of DOK6 and further explored its clinical implication in de novo acute myeloid leukemia (AML).MethodsA total of 100 newly diagnosed adult AML patients were involved in the current study. DOK6 expression and methylation were detected by real‐time qPCR and methylation‐specific PCR (MSP), respectively. Bisulfite sequencing PCR (BSP) was performed to assess the methylation density of the DOK6 promoter.ResultsDownstream of tyrosine kinase 6 promoter methylation was significantly increased in AML patients compared to controls (P = .037), whereas DOK6 expression significantly decreased in AML patients (P < .001). The expression of DOK6 was markedly up‐regulated after treated by 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) in THP‐1 cell lines. The methylation status of the DOK6 promoter was associated with French‐American‐British classifications (P = .037). There was no significant correlation existed between DOK6 expression and its promoter methylation (R = .077, P = .635). Interestingly, of whole‐AML and non‐APL AML patients, both have a tendency pertaining to the DOK6 methylation group and a significantly longer overall survival (OS) than the DOK6 unmethylation group (P = .042 and .036, respectively).ConclusionOur study suggested that DOK6 promoter hypermethylation was a common molecular event in de novo AML patients. Remarkably, DOK6 promoter methylation could serve as an independent and integrated prognostic biomarker not only in non‐APL AML patients but also in AML patients who are less than 60 years old.

Identification of Aberrantly Methylated and Silenced Genes in Acute Myeloid Leukemia by Combined Methylation/expression Analysis

“…Deneberg et al reported that a high level of global methylation predicted a poorer complete remission rate and that p15 methylation was associated with better OS and disease-free survival (DFS) (35). Sun GK et al reported that methylation of the DOK6 promoter predicts longer survival (36). These studies suggested that the methylation level of genes can be valuable prognostic biomarkers.…”

Dysregulation of LINC00324 associated with methylation facilitates leukemogenesis in de novo acute myeloid leukemia

“…Total RNA isolation was conducted as reported previously. 24 Reverse transcription reaction with 40 μl volume was composed of 5× buffer 10 mM, 10 mM of dNTPs (deoxyribonucleoside triphosphates), 10 μM of random hexamers, 80 U of RNAsin, and 200 U of MMLV reverse transcriptase (Eppendorf). The reaction conditions were incubated for 10 min at 25℃, 60 min at 42℃, and then stored at −20℃.…”

Overexpression of lncRNA ITGB2-AS1 Predicts Adverse Prognosis in Acute Myeloid Leukemia

“…Total RNA was extracted from BMNC using Trizol reagent (Invitrogen Life Technologies, USA) as described previously [13][14][15][16][17]. 2 ug of total RNA were reverse transcribed into cDNA using 10 umol/l random primers, 200 U MMLV reverse transcriptase, 0.5 mmol/l dNTP, 10 mmol/l dithiothreitol, and 25 U RNase inhibitors.…”

“…The primers for ITGB2 expression were 5′-GATGACGGCTTCCATTTCGC -3′ (forward) and 5′-TGGGGATGATCTCGGTGAGT -3′ (reverse). Reaction conditions and PCR cycling were conducted as previously described [13][14][15][16][17] except for the optimized primer annealing temperatures (60 °C). The relative quanti cation was calculated using the ΔΔCT method and normalized to the ABL1 housekeeping gene.…”

“…By conventional R-banding method, karyotype was analyzed at the time of initial diagnosis. Risk classi cation based on the karyotype ndings has been done as previously described [13][14][15][16][17]. Mutations in C-KIT, NPM1, DNMT3A, N/K-RAS, and U2AF1 were detected by high-resolution melting analysis, whereas FLT3-ITD and CEBPA mutations were detected by direct DNA sequencing.…”