The acute myeloid leukemia associated AML1-ETO fusion protein alters the transcriptome and cellular progression in a single-oncogene expressing in vitro induced pluripotent stem cell based granulocyte differentiation model

Autoři: Esther Tijchon aff001;  Guoqiang Yi aff001;  Amit Mandoli aff001;  Jos G. A. Smits aff001;  Francesco Ferrari aff001;  Branco M. H. Heuts aff001;  Falco Wijnen aff001;  Bowon Kim aff001;  Eva M. Janssen-Megens aff001;  Jan Jacob Schuringa aff002;  Joost H. A. Martens aff001
Působiště autorů: Radboud University, Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Nijmegen, the Netherlands aff001;  Department of Hematology, University Medical Centre Groningen, Groningen, The Netherlands aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease.

Klíčová slova:

Acute myeloid leukemia – Cell differentiation – Flow cytometry – Gene expression – Granulocytes – Induced pluripotent stem cells – Leukemias – Monocytes


1. Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y, Ohki M. t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. Proceedings of the National Academy of Sciences of the United States of America. 1991;88(23):10431–4. doi: 10.1073/pnas.88.23.10431 1720541

2. Peterson LF, Zhang DE. The 8;21 translocation in leukemogenesis. Oncogene. 2004;23(24):4255–62. doi: 10.1038/sj.onc.1207727 15156181

3. Wang YY, Zhou GB, Yin T, Chen B, Shi JY, Liang WX, et al. AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(4):1104–9. doi: 10.1073/pnas.0408831102 15650049

4. Rhoades KL, Hetherington CJ, Harakawa N, Yergeau DA, Zhou L, Liu LQ, et al. Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model. Blood. 2000;96(6):2108–15. 10979955

5. Yan M, Kanbe E, Peterson LF, Boyapati A, Miao Y, Wang Y, et al. A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis. Nature medicine. 2006;12(8):945–9. doi: 10.1038/nm1443 16892037

6. Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Harakawa N, Hetherington CJ, et al. AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(18):10398–403. doi: 10.1073/pnas.171321298 11526243

7. Peterson LF, Boyapati A, Ahn EY, Biggs JR, Okumura AJ, Lo MC, et al. Acute myeloid leukemia with the 8q22;21q22 translocation: secondary mutational events and alternative t(8;21) transcripts. Blood. 2007;110(3):799–805. doi: 10.1182/blood-2006-11-019265 17412887

8. Nimer SD, Moore MA. Effects of the leukemia-associated AML1-ETO protein on hematopoietic stem and progenitor cells. Oncogene. 2004;23(24):4249–54. doi: 10.1038/sj.onc.1207673 15156180

9. Tonks A, Tonks AJ, Pearn L, Pearce L, Hoy T, Couzens S, et al. Expression of AML1-ETO in human myelomonocytic cells selectively inhibits granulocytic differentiation and promotes their self-renewal. Leukemia. 2004;18(7):1238–45. doi: 10.1038/sj.leu.2403396 15152269

10. Cameron ER, Neil JC. The Runx genes: lineage-specific oncogenes and tumor suppressors. Oncogene. 2004;23(24):4308–14. doi: 10.1038/sj.onc.1207130 15156187

11. de Bruijn MF, Speck NA. Core-binding factors in hematopoiesis and immune function. Oncogene. 2004;23(24):4238–48. doi: 10.1038/sj.onc.1207763 15156179

12. Davis JN, McGhee L, Meyers S. The ETO (MTG8) gene family. Gene. 2003;303:1–10. doi: 10.1016/s0378-1119(02)01172-1 12559562

13. Wang J, Hoshino T, Redner RL, Kajigaya S, Liu JM. ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(18):10860–5. 9724795

14. Sun XJ, Wang Z, Wang L, Jiang Y, Kost N, Soong TD, et al. A stable transcription factor complex nucleated by oligomeric AML1-ETO controls leukaemogenesis. Nature. 2013;500(7460):93–7. doi: 10.1038/nature12287 23812588

15. Wang L, Gural A, Sun XJ, Zhao X, Perna F, Huang G, et al. The leukemogenicity of AML1-ETO is dependent on site-specific lysine acetylation. Science. 2011;333(6043):765–9. doi: 10.1126/science.1201662 21764752

16. Lin S, Ptasinska A, Chen X, Shrestha M, Assi SA, Chin PS, et al. A FOXO1-induced oncogenic network defines the AML1-ETO preleukemic program. Blood. 2017;130(10):1213–22. doi: 10.1182/blood-2016-11-750976 28710059

17. Zhou F, Liu Y, Rohde C, Pauli C, Gerloff D, Kohn M, et al. AML1-ETO requires enhanced C/D box snoRNA/RNP formation to induce self-renewal and leukaemia. Nature cell biology. 2017;19(7):844–55. doi: 10.1038/ncb3563 28650479

18. Mandoli A, Singh AA, Prange KHM, Tijchon E, Oerlemans M, Dirks R, et al. The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs. Cell Rep. 2016;17(8):2087–100. doi: 10.1016/j.celrep.2016.08.082 27851970

19. Schmidl C, Rendeiro AF, Sheffield NC, Bock C. ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors. Nature methods. 2015;12(10):963–5. doi: 10.1038/nmeth.3542 26280331

20. Hansen M, Varga E, Wust T, Brouwer N, Beauchemin H, Mellink C, et al. Generation and characterization of human iPSC line MML-6838-Cl2 from mobilized peripheral blood derived megakaryoblasts. Stem cell research. 2017;18:26–8. doi: 10.1016/j.scr.2016.12.004 28395797

21. Morishima T, Watanabe K, Niwa A, Hirai H, Saida S, Tanaka T, et al. Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis. Haematologica. 2014;99(1):19–27. doi: 10.3324/haematol.2013.083873 23975175

22. Niwa A, Heike T, Umeda K, Oshima K, Kato I, Sakai H, et al. A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors. PloS one. 2011;6(7):e22261. doi: 10.1371/journal.pone.0022261 21818303

23. Marei HE, Althani A, Lashen S, Cenciarelli C, Hasan A. Genetically unmatched human iPSC and ESC exhibit equivalent gene expression and neuronal differentiation potential. Scientific reports. 2017;7(1):17504. doi: 10.1038/s41598-017-17882-1 29235536

24. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based analysis of ChIP-Seq (MACS). Genome biology. 2008;9(9):R137. doi: 10.1186/gb-2008-9-9-r137 18798982

25. Goveia J, Stapor P, Carmeliet P. Principles of targeting endothelial cell metabolism to treat angiogenesis and endothelial cell dysfunction in disease. EMBO molecular medicine. 2014;6(9):1105–20. doi: 10.15252/emmm.201404156 25063693

26. Dicke C, Amirkhosravi A, Spath B, Jimenez-Alcazar M, Fuchs T, Davila M, et al. Tissue factor-dependent and -independent pathways of systemic coagulation activation in acute myeloid leukemia: a single-center cohort study. Experimental hematology & oncology. 2015;4:22.

27. Hussong JW, Rodgers GM, Shami PJ. Evidence of increased angiogenesis in patients with acute myeloid leukemia. Blood. 2000;95(1):309–13. 10607717

28. Litwin C, Leong KG, Zapf R, Sutherland H, Naiman SC, Karsan A. Role of the microenvironment in promoting angiogenesis in acute myeloid leukemia. American journal of hematology. 2002;70(1):22–30. doi: 10.1002/ajh.10092 11994978

29. Trujillo A, McGee C, Cogle CR. Angiogenesis in acute myeloid leukemia and opportunities for novel therapies. Journal of oncology. 2012;2012:128608. doi: 10.1155/2012/128608 21904549

30. Corsello SM, Roti G, Ross KN, Chow KT, Galinsky I, DeAngelo DJ, et al. Identification of AML1-ETO modulators by chemical genomics. Blood. 2009;113(24):6193–205. doi: 10.1182/blood-2008-07-166090 19377049

31. Martens JH, Mandoli A, Simmer F, Wierenga BJ, Saeed S, Singh AA, et al. ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia. Blood. 2012;120(19):4038–48. doi: 10.1182/blood-2012-05-429050 22983443

32. Maiques-Diaz A, Chou FS, Wunderlich M, Gomez-Lopez G, Jacinto FV, Rodriguez-Perales S, et al. Chromatin modifications induced by the AML1-ETO fusion protein reversibly silence its genomic targets through AML1 and Sp1 binding motifs. Leukemia. 2012;26(6):1329–37. doi: 10.1038/leu.2011.376 22289984

33. Steffen B, Knop M, Bergholz U, Vakhrusheva O, Rode M, Kohler G, et al. AML1/ETO induces self-renewal in hematopoietic progenitor cells via the Groucho-related amino-terminal AES protein. Blood. 2011;117(16):4328–37. doi: 10.1182/blood-2009-09-242545 21245488

34. Scholl C, Gilliland DG, Frohling S. Deregulation of signaling pathways in acute myeloid leukemia. Seminars in oncology. 2008;35(4):336–45. doi: 10.1053/j.seminoncol.2008.04.004 18692684

35. Wang YH, Israelsen WJ, Lee D, Yu VWC, Jeanson NT, Clish CB, et al. Cell-state-specific metabolic dependency in hematopoiesis and leukemogenesis. Cell. 2014;158(6):1309–23. doi: 10.1016/j.cell.2014.07.048 25215489

36. Ye M, Zhang H, Yang H, Koche R, Staber PB, Cusan M, et al. Hematopoietic Differentiation Is Required for Initiation of Acute Myeloid Leukemia. Cell stem cell. 2015;17(5):611–23. doi: 10.1016/j.stem.2015.08.011 26412561

37. Ramirez F, Ryan DP, Gruning B, Bhardwaj V, Kilpert F, Richter AS, et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic acids research. 2016;44(W1):W160–5. doi: 10.1093/nar/gkw257 27079975

38. Saia M, Termanini A, Rizzi N, Mazza M, Barbieri E, Valli D, et al. AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells. Scientific reports. 2016;6:34957. doi: 10.1038/srep34957 27713544

39. Lambert SA, Jolma A, Campitelli LF, Das PK, Yin Y, Albu M, et al. The Human Transcription Factors. Cell. 2018;172(4):650–65. doi: 10.1016/j.cell.2018.01.029 29425488

40. Fu L, Huang W, Jing Y, Jiang M, Zhao Y, Shi J, et al. AML1-ETO triggers epigenetic activation of early growth response gene l, inducing apoptosis in t(8;21) acute myeloid leukemia. The FEBS journal. 2014;281(4):1123–31. doi: 10.1111/febs.12673 24314118

41. Hatlen MA, Wang L, Nimer SD. AML1-ETO driven acute leukemia: insights into pathogenesis and potential therapeutic approaches. Frontiers of medicine. 2012;6(3):248–62. doi: 10.1007/s11684-012-0206-6 22875638

42. Arber DA, Slovak ML, Popplewell L, Bedell V, Ikle D, Rowley JD, et al. Therapy-related acute myeloid leukemia/myelodysplasia with balanced 21q22 translocations. American journal of clinical pathology. 2002;117(2):306–13. doi: 10.1309/C3G2-CXA0-HE9J-TKDR 11863228

43. Friedman AD. Leukemogenesis by CBF oncoproteins. Leukemia. 1999;13(12):1932–42. doi: 10.1038/sj.leu.2401590 10602413

44. Lim WF, Inoue-Yokoo T, Tan KS, Lai MI, Sugiyama D. Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells. Stem cell research & therapy. 2013;4(3):71.

45. Wiemels JL, Xiao Z, Buffler PA, Maia AT, Ma X, Dicks BM, et al. In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood. 2002;99(10):3801–5. 11986239

46. Lancrin C, Sroczynska P, Stephenson C, Allen T, Kouskoff V, Lacaud G. The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage. Nature. 2009;457(7231):892–5. doi: 10.1038/nature07679 19182774

47. Thiel VN, Giaimo BD, Schwarz P, Soller K, Vas V, Bartkuhn M, et al. Heterodimerization of AML1/ETO with CBFbeta is required for leukemogenesis but not for myeloproliferation. Leukemia. 2017;31(11):2491–502. doi: 10.1038/leu.2017.105 28360416

48. Klampfer L, Zhang J, Zelenetz AO, Uchida H, Nimer SD. The AML1/ETO fusion protein activates transcription of BCL-2. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(24):14059–64. doi: 10.1073/pnas.93.24.14059 8943060

49. Mulloy J.C., Mackenzie K.L., Berguido F.J., Moore M. a S., and Nimer S.D. The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells. Blood. 2002; 99, 15–23. doi: 10.1182/blood.v99.1.15 11756147

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden