Low-dose decitabine promotes megakaryocyte maturation and platelet production in healthy controls and immune thrombocytopenia

Summary

Impaired megakaryocyte maturation and insufficient platelet production have been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Our previous study demonstrated that low expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in megakaryocytes contributed to impaired platelet production in ITP. Decitabine (DAC), a demethylating agent, is known to promote cell differentiation and maturation at low doses. However, whether decitabine is potential in promoting megakaryocyte maturation and platelet release in ITP is unclear. In this study, we evaluated the effect of DAC on megakaryocyte maturation and platelet release in the presence of ITP plasma that has been shown to cause impaired megakaryocyte maturation and platelet production. We observed that low-dose DAC (10 nM) could significantly increase the number of mature polyploid (≥ 4N) megakaryocytes in cultures with plasma from healthy controls and more than one-half of ITP patients in vitro. Furthermore, the number of platelets released from these megakaryocytes significantly increased compared with those untreated with DAC. In these megakaryocytes, DAC significantly enhanced TRAIL expression via decreasing its promoter methylation status. These findings demonstrate that low-dose DAC can promote megakaryocyte maturation and platelet production and enhance TRAIL expression in megakaryocytes in healthy controls and ITP. The potential therapeutic role of low-dose DAC may be beneficial for thrombocytopenic disorders.

H. Z. and Y. H. contributed equally to this work.

Keywords

Immune thrombocytopenia - decitabine - megakaryocyte - platelet - TRAIL

• References

• 1 Berchtold P, Wenger M. Autoantibodies against platelet glycoproteins in autoimmune thrombocytopenic purpura: their clinical significance and response to treatment. Blood 1993; 81: 1246-1250.
• 2 Alimardani G, Guichard J, Fichelson S. et al. Pathogenic effects of anti-glycoprotein Ib antibodies on megakaryocytes and platelets. Thromb Haemost 2002; 88: 1039-1046.
• 3 Olsson B, Andersson PO, Jernas M. et al. T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nature Med 2003; 09: 1123-1124.
• 4 Stasi R, Evangelista ML, Stipa E. et al. Idiopathic thrombocytopenic purpura: current concepts in pathophysiology and management. Thromb Haemost 2008; 99: 4-13.
• 5 Chang M, Nakagawa PA, Williams SA. et al. Immune thrombocytopenic purpura (ITP) plasma and purified ITP monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro. Blood 2003; 102: 887-895.
• 6 McMillan R, Wang L, Tomer A. et al. Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP. Blood 2004; 103: 1364-1369.
• 7 Li S, Wang L, Zhao C. et al. CD8+ T cells suppress autologous megakaryocyte apoptosis in idiopathic thrombocytopenic purpura. Br J Haematol 2007; 139: 605-611.
• 8 Cines DB, Bussel JB, Liebman HA. et al. The ITP syndrome: pathogenic and clinical diversity. Blood 2009; 113: 6511-6521.
• 9 Ballestar E. Epigenetic alterations in autoimmune rheumatic diseases. Nature Rev Rheumatol 2011; 07: 263-271.
• 10 Heyn H, Esteller M. DNA methylation profiling in the clinic: applications and challenges. Nature Rev Genet 2012; 13: 679-692.
• 11 Zhang Y, Zhao M, Sawalha AH. et al. Impaired DNA methylation and its mechanisms in CD4(+)T cells of systemic lupus erythematosus. J Autoimmun 2013; 41: 92-99.
• 12 Tao J, Yang M, Chen Z. et al. Decreased DNA methyltransferase 3A and 3B mRNA expression in peripheral blood mononuclear cells and increased plasma SAH concentration in adult patients with idiopathic thrombocytopenic purpura. J Clin Immunol 2008; 28: 432-439.
• 13 Ma L, Zhou Z, Wang H. et al. Increased expressions of DNA methyltransferases contribute to CD70 promoter hypomethylation and over expression of CD70 in ITP. Mol Immunol 2011; 48: 1525-1531.
• 14 Li H, Xuan M, Yang R. DNA methylation and primary immune thrombocytopenia. Semin Hematol 2013; 50 (Suppl. 01) S116-S126.
• 15 Kanaji S, Kanaji T, Jacquelin B. et al. Thrombopoietin initiates demethylationbased transcription of GP6 during megakaryocyte differentiation. Blood 2005; 105: 3888-3892.
• 16 Teofili L, Martini M, Di Mario A. et al. Expression of p15(ink4b) gene during megakaryocytic differentiation of normal and myelodysplastic hematopoietic progenitors. Blood 2001; 98: 495-497.
• 17 van den Bosch J, Lubbert M, Verhoef G. et al. The effects of 5-aza-2’-deoxycytidine (Decitabine) on the platelet count in patients with intermediate and highrisk myelodysplastic syndromes. Leukemia Res 2004; 28: 785-790.
• 18 Momparler RL. Pharmacology of 5-Aza-2’-deoxycytidine (decitabine). Semin Hematol 2005; 42 (03) (Suppl. 02) S9-16.
• 19 Wang J, Yi Z, Wang S. et al. The effect of decitabine on megakaryocyte maturation and platelet release. Thromb Haemost 2011; 106: 337-343.
• 20 Yang L, Wang L, Zhao CH. et al. Contributions of TRAIL-mediated megakaryocyte apoptosis to impaired megakaryocyte and platelet production in immune thrombocytopenia. Blood 2010; 116: 4307-4316.
• 21 Zamai L, Secchiero P, Pierpaoli S. et al. TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis. Blood 2000; 95: 3716-3724.
• 22 Crist SA, Elzey BD, Ludwig AT. et al. Expression of TNF-related apoptosis-inducing ligand (TRAIL) in megakaryocytes and platelets. Exp Hematol 2004; 32: 1073-1081.
• 23 Zauli G, Secchiero P. The role of the TRAIL/TRAIL receptors system in hematopoiesis and endothelial cell biology. Cytokin Growth Factor Rev 2006; 17: 245-257.
• 24 Rodeghiero F, Stasi R, Gernsheimer T. et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009; 113: 2386-2393.
• 25 Norol F, Vitrat N, Cramer E. et al. Effects of cytokines on platelet production from blood and marrow CD34+ cells. Blood 1998; 91: 830-843.
• 26 Di Croce L, Raker VA, Corsaro M. et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 2002; 295: 1079-1082.
• 27 Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002; 18: 1427-1431.
• 28 Melloni E, Secchiero P, Celeghini C. et al. Functional expression of TRAIL and TRAIL-R2 during human megakaryocytic development. J Cell Physiol 2005; 204: 975-982.
• 29 Gobbi G, Mirandola P, Sponzilli I. et al. Timing and expression level of protein kinase C epsilon regulate the megakaryocytic differentiation of human CD34 cells. Stem Cells 2007; 25: 2322-2329.
• 30 Derissen EJ, Beijnen JH, Schellens JH. Concise drug review: azacitidine and decitabine. Oncologist 2013; 18: 619-624.
• 31 Steensma DP. Myelodysplasia, megakaryocytes, and methylation. Leukemia Res 2004; 28: 775-776.
• 32 Visvader J, Adams JM. Megakaryocytic differentiation induced in 416B myeloid cells by GATA-2 and GATA-3 transgenes or 5-azacytidine is tightly coupled to GATA-1 expression. Blood 1993; 82: 1493-1501.
• 33 Shivdasani RA, Orkin SH. The transcriptional control of hematopoiesis. Blood 1996; 87: 4025-4039.
• 34 Akashi K, He X, Chen J. et al. Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis. Blood 2003; 101: 383-389.
• 35 de Vos D, van Overveld W. Decitabine: a historical review of the development of an epigenetic drug. Ann Hematol 2005; 84 (Suppl. 01) 3-8.
• 36 Saunthararajah Y, Hillery CA, Lavelle D. et al. Effects of 5-aza-2’-deoxycytidine on fetal hemoglobin levels, red cell adhesion, and hematopoietic differentiation in patients with sickle cell disease. Blood 2003; 102: 3865-3870.
• 37 Olivieri NF, Saunthararajah Y, Thayalasuthan V. et al. A pilot study of subcutaneous decitabine in beta-thalassemia intermedia. Blood 2011; 118: 2708-2711.
• 38 Lubbert M, Suciu S, Baila L. et al. Low-dose decitabine versus best supportive care in elderly patients with intermediate-or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy: final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. J Clin Oncol 2011; 29: 1987-1996.
• 39 Mahfouz RZ, Rickki E, Juersivich JA. et al. Non-Cytotoxic Differentiation Therapy Based On Mechanism of Disease Produces Complete Remission in Myelodysplastic Syndromes (MDS) with High Risk Cytogenetics [abstract]. Blood (ASH Annual Meeting Abstracts) 2012; 120: 1696.
• 40 Phillips CL, Davies SM, McMasters R. et al. Low dose decitabine in very high risk relapsed or refractory acute myeloid leukaemia in children and young adults. Br J Haematol 2013; 161: 406-410.
• 41 Chen ZP, Gu DS, Zhou ZP. et al. Decreased expression of MBD2 and MBD4 gene and genomic-wide hypomethylation in patients with primary immune thrombocytopenia. Human Immunol 2011; 72: 486-491.
• 42 El-Shiekh EH, Bessa SS, Abdou SM. et al. Role of DNA methyltransferase 3A mRNA expression in Egyptian patients with idiopathic thrombocytopenic purpura. Intern J Lab Hematol 2012; 34: 369-376.
• 43 De Botton S, Sabri S, Daugas E. et al. Platelet formation is the consequence of caspase activation within megakaryocytes. Blood 2002; 100: 1310-1317.
• 44 Clarke MC, Savill J, Jones DB. et al. Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death. J Cell Biol 2003; 160: 577-587.
• 45 Kaluzhny Y, Yu G, Sun S. et al. BclxL overexpression in megakaryocytes leads to impaired platelet fragmentation. Blood 2002; 100: 1670-1678.
• 46 White MJ, Schoenwaelder SM, Josefsson EC. et al. Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function. Blood 2012; 119: 4283-4290.
• 47 Josefsson EC, Burnett DL, Lebois M. et al. Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways. Nature Commun 2014; 05: 3455.
• 48 Attwood JT, Yung RL, Richardson BC. DNA methylation and the regulation of gene transcription. Cell Mol Life Sci 2002; 59: 241-257.
• 49 Paulsen M, Ferguson-Smith AC. DNA methylation in genomic imprinting, development, and disease. J Pathol 2001; 195: 97-110.
• 50 Soncini M, Santoro F, Gutierrez A. et al. The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia. Biochim Biophys Acta 2013; 1832: 114-120.