Tumor Biology

, Volume 37, Issue 7, pp 8951–8960 | Cite as

DLX4 hypermethylation is a prognostically adverse indicator in de novo acute myeloid leukemia

  • Jing-dong Zhou
  • Ting-juan Zhang
  • Yu-xin Wang
  • Dong-qin Yang
  • Lei Yang
  • Ji-chun Ma
  • Xiang-mei Wen
  • Jing Yang
  • Jiang Lin
  • Jun Qian
Original Article

Abstract

Hypermethylation of distal-less homeobox 4 (DLX4) has been increasingly identified in several cancers. Our study was aimed to determine the role of DLX4 methylation in regulating DLX4 expression and further analyze its clinical significance in de novo acute myeloid leukemia (AML) patients. DLX4 methylation level was detected by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Treatment with 5-aza-2′-deoxycytidine (5-aza-dC) was used for demethylation studies. Clinical significance of DLX4 methylation was obtained by the comparison between the patients with and without DLX4 methylation. DLX4 was significantly methylated in AML patients compared with controls (P < 0.001). DLX4 methylation was negatively associated with DLX7 (the shorter DLX4 isoform) (R = −0.202, P = 0.021) but not BP1 (the longer DLX4 isoform) (R = −0.049, P = 0.582) expression in AML patients. DLX7 and BP1 messenger RNA (mRNA) were significantly increased after 5-aza-dC treatment in leukemic cell lines THP1 and Kasumi-1. DLX4 methylated patients showed significantly higher frequency of U2AF1 mutation compared with DLX4 unmethylated patients (P = 0.043). Both all AML and non-M3 patients with DLX4 methylation presented significantly lower complete remission rate than those with DLX4 unmethylation (P = 0.001 and <0.001, respectively). DLX4 methylated cases had significantly shorter overall survival than DLX4 unmethylated cases among both all AML (P = 0.003), non-M3 AML (P = 0.001), and cytogenetically normal AML (P = 0.032). Multivariate analysis confirmed that DLX4 methylation was independent risk factor in both all AML and non-M3 patients. Our study indicates that DLX4 hypermethylation is negatively associated with DLX7 expression and predicts poor clinical outcome in de novo AML patients.

Keywords

DLX4 Methylation Expression Prognosis AML 

Notes

Acknowledgments

This study was supported by National Natural Science Foundation of China (81270630, 81172592), Science and Technology Special Project in Clinical Medicine of Jiangsu Province (BL2012056), 333 Project of Jiangsu Province (BRA2013136), Science and Technology Infrastructure Program of Zhenjiang (SS2012003), and Medical Key Talent Project of Zhenjiang, Social Development Foundation of Zhenjiang (SH2013042, SH2013082, SH2014044, SH2014086).

Compliance with ethical standards

Conflicts of interest

None.

References

  1. 1.
    Estey E, Döhmer H. Acute myeloid leukaemia. Lancet. 2006;368:1894–907.CrossRefPubMedGoogle Scholar
  2. 2.
    Lane SW, Scadden DT, Gilliland DG. The leukemic stem cell niche: current concepts and therapeutic opportunities. Blood. 2009;114:1150–7.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Avivi I, Rowe JM. Prognostic factors in acute myeloid leukemia. Curr Opin Hematol. 2005;12:62–7.CrossRefPubMedGoogle Scholar
  4. 4.
    Marcucci G, Haferlach T, Döhner H. Molecular genetics of adult acute myeloid leukemia: prognostic and therapeutic implications. J Clin Oncol. 2011;29:475–86.CrossRefPubMedGoogle Scholar
  5. 5.
    Marchesi F, Annibali O, Cerchiara E, Tirindelli MC, Avvisati G. Cytogenetic abnormalities in adult non-promyelocytic acute myeloid leukemia: a concise review. Crit Rev Oncol Hematol. 2011;80:331–46.CrossRefPubMedGoogle Scholar
  6. 6.
    Grimwade D. The clinical significance of cytogenetic abnormalities in acute myeloid leukaemia. Best Pract Res Clin Haematol. 2001;14:497–529.CrossRefPubMedGoogle Scholar
  7. 7.
    Byrd JC, Mrózek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002;100:4325–36.CrossRefPubMedGoogle Scholar
  8. 8.
    Abate-Shen C. Deregulated homeobox gene expression in cancer: cause or consequence? Nat Rev Cancer. 2002;2:777–85.CrossRefPubMedGoogle Scholar
  9. 9.
    Kraus P, Lufkin T. Dlx homeobox gene control of mammalian limb and craniofacial development. Am J Med Genet A. 2006;140:1366–74.CrossRefPubMedGoogle Scholar
  10. 10.
    Mpollo MS, Beaudoin M, Berg PE, Beauchemin H, D’Agati V, Trudel M. BP1 is a negative modulator of definitive erythropoiesis. Nucleic Acids Res. 2006;34:5232–7.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tomida S, Yanagisawa K, Koshikawa K, Yatabe Y, Mitsudomi T, Osada H, et al. Identification of a metastasis signature and the DLX4 homeobox protein as a regulator of metastasis by combined transcriptome approach. Oncogene. 2007;26:4600–8.CrossRefPubMedGoogle Scholar
  12. 12.
    Trinh BQ, Barengo N, Naora H. Homeodomain protein DLX4 counteracts key transcriptional control mechanisms of the TGF-β cytostatic program and blocks the antiproliferative effect of TGF-β. Oncogene. 2011;30:2718–29.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhang L, Yang M, Gan L, He T, Xiao X, Stewart MD, et al. DLX4 upregulates TWIST and enhances tumor migration, invasion and metastasis. Int J Biol Sci. 2012;8:1178–87.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Sun Y, Lu X, Yin L, Zhao F, Feng Y. Inhibition of DLX4 promotes apoptosis in choriocarcinoma cell lines. Placenta. 2006;27:375–83.CrossRefPubMedGoogle Scholar
  15. 15.
    Miyamoto K, Fukutomi T, Akashi-Tanaka S, Hasegawa T, Asahara T, Sugimura T, et al. Identification of 20 genes aberrantly methylated in human breast cancers. Int J Cancer. 2005;116:407–14.CrossRefPubMedGoogle Scholar
  16. 16.
    Tong WG, Wierda WG, Lin E, Kuang SQ, Bekele BN, Estrov Z, et al. Genome-wide DNA methylation profiling of chronic lymphocytic leukemia allows identification of epigenetically repressed molecular pathways with clinical impact. Epigenetics. 2010;5:499–508.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Rauch TA, Wang Z, Wu X, Kernstine KH, Riggs AD, Pfeifer GP. DNA methylation biomarkers for lung cancer. Tumor Biol. 2012;33:287–96.CrossRefGoogle Scholar
  18. 18.
    Sakane J, Taniyama K, Miyamoto K, Saito A, Kuraoka K, Nishimura T, et al. Aberrant DNA methylation of DLX4 and SIM1 is a predictive marker for disease progression of uterine cervical low-grade squamous intraepithelial lesion. Diagn Cytopathol. 2015;43:462–70.CrossRefPubMedGoogle Scholar
  19. 19.
    Harada H, Miyamoto K, Yamashita Y, Taniyama K, Ohdan H, Okada M. Methylated DLX4 predicts response to pathologic stage I non-small cell lung cancer resection. Ann Thorac Surg. 2015;99:1746–54.CrossRefPubMedGoogle Scholar
  20. 20.
    Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2008.Google Scholar
  21. 21.
    Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposed revised criteria for the classification of acute myeloid leukaemia. A report of the French-American-British Cooperative Group. Ann Intern Med. 1985;103:620–5.CrossRefPubMedGoogle Scholar
  22. 22.
    Grimwade D, Hills RK, Moorman AV, Walker H, Chatters S, Goldstone AH, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood. 2010;116:354–65.CrossRefPubMedGoogle Scholar
  23. 23.
    Li Y, Lin J, Yang J, Qian J, Qian W, Yao DM, et al. Overexpressed let-7a-3 is associated with poor outcome in acute myeloid leukemia. Leuk Res. 2013;37:1642–7.CrossRefPubMedGoogle Scholar
  24. 24.
    Zhou JD, Yang L, Zhang YY, Yang J, Wen XM, Guo H, et al. Overexpression of BAALC: clinical significance in Chinese de novo acute myeloid leukemia. Med Oncol. 2015;32:386.CrossRefPubMedGoogle Scholar
  25. 25.
    Lin J, Yao DM, Qian J, Chen Q, Qian W, Li Y, et al. Recurrent DNMT3A R882 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome. PLoS One. 2011;6:e26906.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lin J, Yao DM, Qian J, Chen Q, Qian W, Li Y, et al. IDH1 and IDH2 mutation analysis in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome. Ann Hematol. 2012;91:519–25.CrossRefPubMedGoogle Scholar
  27. 27.
    Yang X, Qian J, Sun A, Lin J, Xiao G, Yin J, et al. RAS mutation analysis in a large cohort of Chinese patients with acute myeloid leukemia. Clin Biochem. 2013;46:579–83.CrossRefPubMedGoogle Scholar
  28. 28.
    Qian J, Yao DM, Lin J, Qian W, Wang CZ, Chai HY, et al. U2AF1 mutations in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome. PLoS One. 2012;7:e45760.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Wen XM, Lin J, Yang J, Yao DM, Deng ZQ, Tang CY, et al. Double CEBPA mutations are prognostically favorable in non-M3 acute myeloid leukemia patients with wild-type NPM1 and FLT3-ITD. Int J Clin Exp Pathol. 2014;7:6832–40.PubMedPubMedCentralGoogle Scholar
  30. 30.
    Zhou JD, Wang YX, Zhang TJ, Yang DQ, Yao DM, Guo H, et al. Epigenetic inactivation of DLX4 is associated with disease progression in chronic myeloid leukemia. Biochem Biophys Res Commun. 2015;463:1250–6.CrossRefPubMedGoogle Scholar
  31. 31.
    Zhang TJ, Zhou JD, Yang DQ, Wang YX, Yao DM, Ma JC, et al. Hypermethylation of DLX4 predicts poor clinical outcome in patients with myelodysplastic syndrome. Clin Chem Lab Med. 2015.Google Scholar
  32. 32.
    Oki Y, Issa JP. Epigenetic mechanisms in AML—a target for therapy. Cancer Treat Res. 2010;145:19–40.CrossRefPubMedGoogle Scholar
  33. 33.
    Shaknovich R. Gene expression and epigenetic deregulation. Adv Exp Med Biol. 2013;792:133–50.CrossRefPubMedGoogle Scholar
  34. 34.
    Gao Y, Li Z, Guo X, Liu Y, Zhang K. DLX4 as a prognostic marker for hepatocellular carcinoma. Neoplasma. 2014;61:318–23.CrossRefPubMedGoogle Scholar
  35. 35.
    Yu M, Wan YF, Zou QH. Prognostic significance of BP1 mRNA expression level in patients with non-small cell lung cancer. Clin Biochem. 2008;41:824–30.CrossRefPubMedGoogle Scholar
  36. 36.
    Yu M, Yang Y, Shi Y, Wang D, Wei X, Zhang N, et al. Expression level of beta protein 1 mRNA in Chinese breast cancer patients: a potential molecular marker for poor prognosis. Cancer Sci. 2008;99:173–8.PubMedGoogle Scholar
  37. 37.
    Haga SB, Fu S, Karp JE, Ross DD, Williams DM, Hankins WD, et al. BP1, a new homeobox gene, is frequently expressed in acute leukemias. Leukemia. 2000;14:1867–75.CrossRefPubMedGoogle Scholar
  38. 38.
    Zhou JD, Yang J, Guo H, Deng ZQ, Wen XM, Yang L, et al. BP1 overexpression is associated with adverse prognosis in de novo acute myeloid leukemia. Leuk Lymphoma. 2015:1–19.Google Scholar
  39. 39.
    Xie XH, Xu XP, Sun CY, Yu ZJ. Regulation of the oncogenic function of distal-less 4 by microRNA-122 in hepatocellular carcinoma. Mol Med Rep. 2015;12:1375–80.PubMedGoogle Scholar
  40. 40.
    Woods BA, Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies. Immunol Rev. 2015;263:22–35.CrossRefPubMedGoogle Scholar
  41. 41.
    Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer. 2012;12:599–612.CrossRefPubMedGoogle Scholar
  42. 42.
    Ilagan JO, Ramakrishnan A, Hayes B, Murphy ME, Zebari AS, Bradley P, et al. U2AF1 mutations alter splice site recognition in hematological malignancies. Genome Res. 2015;25:14–26.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Jing-dong Zhou
    • 1
  • Ting-juan Zhang
    • 1
  • Yu-xin Wang
    • 1
  • Dong-qin Yang
    • 1
  • Lei Yang
    • 1
  • Ji-chun Ma
    • 2
  • Xiang-mei Wen
    • 2
  • Jing Yang
    • 1
  • Jiang Lin
    • 2
  • Jun Qian
    • 1
  1. 1.Department of HematologyAffiliated People’s Hospital of Jiangsu UniversityZhenjiangPeople’s Republic of China
  2. 2.Laboratory CenterAffiliated People’s Hospital of Jiangsu UniversityZhenjiangPeople’s Republic of China

Personalised recommendations