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

Guo-Kang Sun, Li-Juan Tang, Jing-Dong Zhou, Zi-Jun Xu, Lan Yang, Qian Yuan, Ji-Chun Ma, Xing-Hui Liu, Jiang Lin, Jun Qian, Dong-Ming Yao, Guo-Kang Sun, Li-Juan Tang, Jing-Dong Zhou, Zi-Jun Xu, Lan Yang, Qian Yuan, Ji-Chun Ma, Xing-Hui Liu, Jiang Lin, Jun Qian, Dong-Ming Yao

Abstract

Background: Downstream 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).

Methods: A 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.

Results: Downstream 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).

Conclusion: Our 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.

Keywords: DOK6; AML; biomarker; methylation; prognosis.

Conflict of interest statement

None declared.

© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Figures

Figure 1
Figure 1
Downstream of tyrosine kinase 6 (DOK6) methylation primer position and methylation density of the DOK6 promoter in AML. A, The genomic coordinates (GC) of DOK6 promoter region CpG island and primer locations. The panel plots the GC content as a percentage of the total. Each vertical bar in the bottom panel represents the presence of a CpG dinucleotide. Black horizontal bars indicate regions amplified by MSP primer pairs and BSP primer pairs. This figure was created using Methyl Primer Express v1.0 software and CpGplot (http://emboss.bioinformatics.nl/cgi-bin/emboss/cpgplot). AML, acute myeloid leukemia; BSP, bisulfite sequencing PCR; MSP, methylation‐specific PCR; TSS, transcription start site. B, Relative promoter methylation level of DOK6 in AML patients and controls. DOK6 methylation level was examined by MSP DOK6 methylation level was up‐regulated in AML patients compare to controls. MSP: methylation‐specific PCR. C, Methylation density of DOK6 promoter in AML patients and controls. Methylation density was determined by BSP. White cycle: unmethylated CpG dinucleotide; Black cycle: methylated CpG dinucleotide. P1: methylated AML patient; P2: unmethylated AML patient; P3 and P4: controls
Figure 2
Figure 2
Downstream of tyrosine kinase 6 (DOK6) expression levels in THP‐1 cell line and acute myeloid leukemia (AML). A, DOK6 relative expression in THP‐1 cell line with or without 5‐aza‐dC treatment; DOK6 expression level was examined by real‐time quantitative PCR (qPCR). B, DOK6 promoter methylation levels in THP‐1 cell line with or without 5‐aza‐dC treatment; DOK6 methylation level was examined by methylation‐specific PCR. C, Relative expression level of DOK6 in AML patients and controls. DOK6 expression level was examined by qPCR
Figure 3
Figure 3
Prognostic value of downstream of tyrosine kinase 6 (DOK6) methylation in acute myeloid leukemia (AML) patients. (A, B, D) For DOK6 methylation in AML patients and non‐APL AML patients as well as AML patients who are less than 60 years old. C, For DOK6 expression in de novo AML patients. DOK6 expression level was examined by real‐time quantitative PCR

References

    1. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373(12):1136‐1152.
    1. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424‐447.
    1. Cancer Genome Atlas Research Network , Ley TJ, Miller C, Ding L, et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368(22):2059–74.
    1. Kim J, Fox C, Peng S, et al. Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2‐amplified gastroesophageal adenocarcinoma. J Clin Invest. 2014;124(12):5145‐5158.
    1. Carpino N, Wisniewski D, Strife A, et al. p62(dok): a constitutively tyrosine‐phosphorylated, GAP‐associated protein in chronic myelogenous leukemia progenitor cells. Cell. 1997;88(2):197‐204.
    1. Yamanashi Y, Baltimore D. Identification of the Abl‐ and rasGAP‐associated 62 kDa protein as a docking protein, Dok. Cell. 1997;88(2):205‐211.
    1. Manning G, Young SL, Miller WT, Zhai Y. The protist, Monosiga brevicollis, has a tyrosine kinase signaling network more elaborate and diverse than found in any known metazoan. Proc Natl Acad Sci USA. 2008;105(28):9674‐9679.
    1. Pincus D, Letunic I, Bork P, Lim WA. Evolution of the phospho‐tyrosine signaling machinery in premetazoan lineages. Proc Natl Acad Sci USA. 2008;105(28):9680‐9684.
    1. Di Cristofano A, Niki M, Zhao M, et al. p62(dok), a negative regulator of Ras and mitogen‐activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr‐abl). J Exp Med. 2001;194(3):275–84.
    1. Gugasyan R, Quilici C, I ST, et al. Dok‐related protein negatively regulates T cell development via its RasGTPase‐activating protein and Nck docking sites. J Cell Biol. 2002;158(1):115‐125.
    1. Yasuda T, Shirakata M, Iwama A, et al. Role of Dok‐1 and Dok‐2 in myeloid homeostasis and suppression of leukemia. J Exp Med. 2004;200(12):1681‐1687.
    1. Tamir I, Stolpa JC, Helgason CD, et al. The RasGAP‐binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells. Immunity. 2000;12:347‐358.
    1. Berger AH, Niki M, Morotti A, et al. Identification of DOK genes as lung tumor suppressors. Nat Genet. 2010;42(3):216‐223.
    1. Mashima R, Honda K, Yang YI, et al. Mice lacking Dok‐1, Dok‐2, and Dok‐3 succumb to aggressive histiocytic sarcoma. Lab Invest. 2010;90(9):1357‐1364.
    1. Grimm J, Sachs M, Britsch S, et al. Novel p62dok family members, dok‐4 and dok‐5, are substrates of the c‐Ret receptor tyrosine kinase and mediate neuronal differentiation. J Cell Biol. 2001;154(2):345‐354.
    1. Cai D, Dhe‐Paganon S, Melendez PA, Lee J, Shoelson SE. Two new substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5. J Biol Chem. 2003;278(28):25323‐25330.
    1. Okada K, Inoue A, Okada M, et al. The muscle protein Dok‐7 is essential for neuromuscular synaptogenesis. Science. 2006;312(5781):1802‐1805.
    1. Crowder RJ, Enomoto H, Yang M, Johnson EM Jr, Milbrandt J. Dok‐6, a Novel p62 Dok family member, promotes Ret‐mediated neurite outgrowth. J Biol Chem. 2004;279(40):42072‐42081.
    1. Kurotsuchi A, Murakumo Y, Jijiwa M, et al. Analysis of DOK‐6 function in downstream signaling of RET in human neuroblastoma cells. Cancer Sci. 2010;101(5):1147‐1155.
    1. Li W, Shi L, You Y, et al. Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin‐related kinase C receptor, is involved in neurotrophin 3‐mediated neurite outgrowth in mouse cortex neurons. BMC Biol. 2010;8:86.
    1. Leong SH, Lwin KM, Lee SS, et al. Chromosomal breaks at FRA18C: association with reduced DOK6 expression, altered oncogenic signaling and increased gastric cancer survival. NPJ Precis Oncol. 2017;1(1):9.
    1. Ghanem T, Bracken J, Kasem A, Jiang WG, Mokbel K. mRNA expression of DOK1‐6 in human breast cancer. World J Clin Oncol. 2014;5(2):156‐163.
    1. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127:2391‐2405.
    1. Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French‐American‐British Cooperative Group. Ann Intern Med. 1985;103(4):620‐625.
    1. Zhou JD, Zhang TJ, Li XX, et al. Epigenetic dysregulation of ID4 predicts disease progression and treatment outcome in myeloid malignancies. J Cell Mol Med. 2017;21(8):1468‐1481.
    1. Zhou JD, Wang YX, Zhang TJ, et al. Identification and validation of SRY‐box containing gene family member SOX30 methylation as a prognostic and predictive biomarker in myeloid malignancies. Clin Epigenetics. 2018;10:92.
    1. Zhang TJ, Zhou JD, Zhang W, et al. H19 overexpression promotes leukemogenesis and predicts unfavorable prognosis in acute myeloid leukemia. Clin Epigenetics. 2018;10:47.
    1. Mashima R, Hishida Y, Tezuka T, Yamanashi Y. The roles of Dok family adapters in immunoreceptor signaling. Immunol Rev. 2009;232(1):273‐285.
    1. Jones N, Dumont DJ. Recruitment of Dok‐R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation. Curr Biol. 1999;9(18):1057‐1060.
    1. Di Cristofano A, Carpino N, Dunant N, et al. Molecular cloning and characterization of p56dok‐2 defines a new family of RasGAP‐binding proteins. J Biol Chem. 1998;273(9):4827‐4830.
    1. Lemay S, Davidson D, Latour S, Veillette A. Dok‐3, a novel adapter molecule involved in the negative regulation of immunoreceptor signaling. Mol Cell Biol. 2000;20(8):2743‐2754.
    1. He PF, Xu ZJ, Zhou JD, et al. Methylation‐associated DOK1 and DOK2 down‐regulation: potential biomarkers for predicting adverse prognosis in acute myeloid leukemia. J Cell Physiol. 2018;233(9):6604‐6614.
    1. Zhang L, Li R, Hu K, et al. Prognostic role of DOK family adapters in acute myeloid leukemia. Cancer Gene Ther. 2018.
    1. Terashima M, Kitada K, Ochiai A, et al. ACTS‐GC Group. Impact of expression of human epidermal growth factor receptors EGFR and ERBB2 on survival in stage II/III gastric cancer. Clin Cancer Res. 2012;18(21):5992‐6000.
    1. Betts G, Valentine H, Pritchard S, et al. FGFR2, HER2 and cMet in gastric adenocarcinoma: detection, prognostic significance and assessment of downstream pathway activation. Virchows Arch. 2014;464(2):145‐156.
    1. Kodama M, Kitadai Y, Sumida T, et al. Expression of platelet‐derived growth factor (PDGF)‐B and PDGF‐receptor β is associated with lymphatic metastasis in human gastric carcinoma. Cancer Sci. 2010;101(9):1984‐1989.
    1. Ford MG, Valle JD, Soroka CJ, Merchant JL. EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures. J Clin Invest. 1997;99(11):2762‐2771.
    1. Taby R, Issa JP. Cancer epigenetics. CA Cancer J Clin. 2010;60(6):376‐392.

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe