Activating mutations in the receptor tyrosine kinase FLT3 are one of the most frequent somatic mutations in acute myeloid leukemia (AML). Internal tandem duplications of the juxtamembrane region of FLT3 (FLT3/ITD) constitutively activate survival and proliferation pathways, and are associated with a poor prognosis in AML. We suspected that alteration of small non-coding microRNA (miRNA) expression in these leukemia cells is involved in the transformation process and used miRNA microarrays to determine the miRNA signature from total RNA harvested from FLT3/ITD expressing FDC-P1 cells (FD-FLT3/ITD). This revealed that a limited set of miRNAs appeared to be affected by expression of FLT3/ITD compared to the control group consisting of FDC-P1 parental cells transfected with an empty vector (FD-EV). Among differentially expressed miRNAs, we selected miR-16, miR-21 and miR-223 to validate the microarray data by quantitative real-time RT-PCR showing a high degree of correlation. We further analyzed miR-16 expression with FLT3 inhibitors in FLT3/ITD expressing cells. MiR-16 was found to be one of most significantly down-regulated miRNAs in FLT3/ITD expressing cells and was up-regulated upon FLT3 inhibition. The data suggests that miR-16 is acting as a tumour suppressor gene in FLT3/ITD-mediated leukemic transformation. Whilst miR-16 has been reported to target multiple mRNAs, computer models from public bioinformatic resources predicted a potential regulatory mechanism between miR-16 and Pim-1 mRNA. In support of this interaction, miR-16 was shown to suppress Pim-1 reporter gene expression. Further, our data demonstrated that over-expression of miR-16 mimics suppressed Pim-1 expression in FD-FLT3/ITD cells suggesting that increased miR-16 expression contributes to depletion of Pim-1 after FLT3 inhibition and that miR-16 repression may be associated with up-regulated Pim-1 in FLT3/ITD expressing cells.
References
[1]
Birg F, Courcoul M, Rosnet O, Bardin F, Pebusque MJ, et al. (1992) Expression of the FMS/KIT-like gene FLT3 in human acute leukemias of the myeloid and lymphoid lineages. Blood 80: 2584–2593.
[2]
Carow CE, Levenstein M, Kaufmann SH, Chen J, Amin S, et al. (1996) Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias. Blood 87: 1089–1096.
[3]
Rosnet O, Buhring HJ, Marchetto S, Rappold I, Lavagna C, et al. (1996) Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia 10: 238–248.
[4]
Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, et al. (2002) Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 99: 4326–4335.
[5]
Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, et al. (1996) Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 10: 1911–1918.
[6]
Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, et al. (1997) Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines. Leukemia 11: 1605–1609.
[7]
Levis M, Small D (2003) FLT3: ITDoes matter in leukemia. Leukemia 17: 1738–1752.
[8]
Kim KT, Baird K, Ahn JY, Meltzer P, Lilly M, et al. (2005) Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival. Blood 105: 1759–1767.
[9]
Kim KT, Baird K, Davis S, Piloto O, Levis M, et al. (2007) Constitutive Fms-like tyrosine kinase 3 activation results in specific changes in gene expression in myeloid leukaemic cells. Br J Haematol 138: 603–615.
[10]
Choudhary C, Brandts C, Schwable J, Tickenbrock L, Sargin B, et al. (2007) Activation mechanisms of STAT5 by oncogenic Flt3-ITD. Blood 110: 370–374.
[11]
Peltola KJ, Paukku K, Aho TL, Ruuska M, Silvennoinen O, et al. (2004) Pim-1 kinase inhibits STAT5-dependent transcription via its interactions with SOCS1 and SOCS3. Blood 103: 3744–3750.
[12]
Vu HA, Xinh PT, Kano Y, Tokunaga K, Sato Y (2009) The juxtamembrane domain in ETV6/FLT3 is critical for PIM-1 up-regulation and cell proliferation. Biochem Biophys Res Commun 383: 308–313.
Carthew RW, Sontheimer EJ (2009) Origins and Mechanisms of miRNAs and siRNAs. Cell 136: 642–655.
[17]
Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, et al. (2005) miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A 102: 13944–13949.
[18]
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435: 834–838.
[19]
Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, et al. (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A 103: 2257–2261.
[20]
Calin GA, Garzon R, Cimmino A, Fabbri M, Croce CM (2006) MicroRNAs and leukemias: how strong is the connection? Leuk Res 30: 653–655.
[21]
Lynam-Lennon N, Maher SG, Reynolds JV (2009) The roles of microRNA in cancer and apoptosis. Biol Rev Camb Philos Soc 84: 55–71.
[22]
Lawrie CH (2007) MicroRNAs and haematology: small molecules, big function. Br J Haematol 137: 503–512.
[23]
Yendamuri S, Calin GA (2009) The role of microRNA in human leukemia: a review. Leukemia 23: 1257–1263.
[24]
Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, et al. (2002) Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 99: 15524–15529.
[25]
Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, et al. (2005) A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 353: 1793–1801.
[26]
Marton S, Garcia MR, Robello C, Persson H, Trajtenberg F, et al. (2008) Small RNAs analysis in CLL reveals a deregulation of miRNA expression and novel miRNA candidates of putative relevance in CLL pathogenesis. Leukemia 22: 330–338.
[27]
Mi S, Lu J, Sun M, Li Z, Zhang H, et al. (2007) MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia. Proc Natl Acad Sci U S A 104: 19971–19976.
[28]
Garzon R, Volinia S, Liu CG, Fernandez-Cymering C, Palumbo T, et al. (2008) MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood 111: 3183–3189.
[29]
Li Z, Lu J, Sun M, Mi S, Zhang H, et al. (2008) Distinct microRNA expression profiles in acute myeloid leukemia with common translocations. Proc Natl Acad Sci U S A 105: 15535–15540.
[30]
Flamant S, Ritchie W, Guilhot J, Holst J, Bonnet ML, et al. (2010) Micro-RNA response to imatinib mesylate in patients with chronic myeloid leukemia. Haematologica 95: 1325–1333.
[31]
Bhattacharya R, Nicoloso M, Arvizo R, Wang E, Cortez A, et al. (2009) MiR-15a and MiR-16 control Bmi-1 expression in ovarian cancer. Cancer Res 69: 9090–9095.
[32]
Kaddar T, Rouault JP, Chien WW, Chebel A, Gadoux M, et al. (2009) Two new miR-16 targets: caprin-1 and HMGA1, proteins implicated in cell proliferation. Biol Cell 101: 511–524.
[33]
Chen RW, Bemis LT, Amato CM, Myint H, Tran H, et al. (2008) Truncation in CCND1 mRNA alters miR-16–1 regulation in mantle cell lymphoma. Blood 112: 822–829.
[34]
Zhang X, Wan G, Mlotshwa S, Vance V, Berger FG, et al. (2010) Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway. Cancer Research 70: 7176–7186.
[35]
Baudry A, Mouillet-Richard S, Schneider B, Launay JM, Kellermann O (2010) miR-16 targets the serotonin transporter: a new facet for adaptive responses to antidepressants. Science 329: 1537–1541.
[36]
Kim KT, Levis M, Small D (2006) Constitutively activated FLT3 phosphorylates BAD partially through pim-1. Br J Haematol 134: 500–509.
[37]
Adam M, Pogacic V, Bendit M, Chappuis R, Nawijn MC, et al. (2006) Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL. Cancer Res 66: 3828–3835.
[38]
Chen LS, Redkar S, Taverna P, Cortes JE, Gandhi V (2011) Mechanisms of cytotoxicity to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia. Blood 118: 693–702.
[39]
Fathi AT, Arowojolu O, Swinnen I, Sato T, Rajkhowa T, et al. (2012) A potential therapeutic target for FLT3-ITD AML: PIM1 kinase. Leuk Res 36: 224–231.
[40]
Grundler R, Brault L, Gasser C, Bullock AN, Dechow T, et al. (2009) Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration. J Exp Med 206: 1957–1970.
[41]
Frost MJ, Ferrao PT, Hughes TP, Ashman LK (2002) Juxtamembrane mutant V560GKit is more sensitive to Imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant. Mol Cancer Ther 1: 1115–1124.
[42]
Roberts KG, Odell AF, Byrnes EM, Baleato RM, Griffith R, et al. (2007) Resistance to c-KIT kinase inhibitors conferred by V654A mutation. Mol Cancer Ther 6: 1159–1166.
[43]
Mashkani B, Griffith R, Ashman LK (2010) Colony stimulating factor-1 receptor as a target for small molecule inhibitors. Bioorg Med Chem 18: 1789–1797.
[44]
Gao X, Gulari E, Zhou X (2004) In situ synthesis of oligonucleotide microarrays. Biopolymers 73: 579–596.
[45]
Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19: 185–193.
[46]
Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 95: 14863–14868.
[47]
Beveridge NJ, Tooney PA, Carroll AP, Tran N, Cairns MJ (2009) Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation. Cell Signal 21: 1837–1845.
[48]
Beveridge NJ, Gardiner E, Carroll AP, Tooney PA, Cairns MJ (2010) Schizophrenia is associated with an increase in cortical microRNA biogenesis. Mol Psychiatry 15: 1176–1189.
[49]
Shounan Y, Miller M, Symonds G (1995) Transformation of FDC-P1 cells to IL-3 independence by a recombinant murine retrovirus containing v-erb-B. Exp Hematol 23: 492–499.
[50]
Young SM, Cambareri AC, Ashman LK (2006) Role of c-KIT expression level and phosphatidylinositol 3-kinase activation in survival and proliferative responses of early myeloid cells. Cell Signal 18: 608–620.
[51]
Griffith J, Black J, Faerman C, Swenson L, Wynn M, et al. (2004) The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. Mol Cell 13: 169–178.
[52]
Tse KF, Allebach J, Levis M, Smith BD, Bohmer FD, et al. (2002) Inhibition of the transforming activity of FLT3 internal tandem duplication mutants from AML patients by a tyrosine kinase inhibitor. Leukemia 16: 2027–2036.
[53]
Zheng R, Friedman AD, Levis M, Li L, Weir EG, et al. (2004) Internal tandem duplication mutation of FLT3 blocks myeloid differentiation through suppression of C/EBPalpha expression. Blood 103: 1883–1890.
[54]
Mizuki M, Fenski R, Halfter H, Matsumura I, Schmidt R, et al. (2000) Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 96: 3907–3914.
[55]
Mizuki M, Schwable J, Steur C, Choudhary C, Agrawal S, et al. (2003) Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations. Blood 101: 3164–3173.
[56]
Levis M, Pham R, Smith BD, Small D (2004) In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects. Blood 104: 1145–1150.
[57]
Smith BD, Levis M, Beran M, Giles F, Kantarjian H, et al. (2004) Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia. Blood 103: 3669–3676.
[58]
O’Farrell AM, Abrams TJ, Yuen HA, Ngai TJ, Louie SG, et al. (2003) SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 101: 3597–3605.
[59]
Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, et al. (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455: 58–63.
[60]
Enjeti AK, Tien SL, Sivaswaren CR (2004) Cytogenetic abnormalities in de novo acute myeloid leukemia in adults: relation to morphology, age, sex and ethnicity - a single center study from Singapore. Hematol J 5: 419–425.
[61]
Georgantas RW, 3rd, Hildreth R, Morisot S, Alder J, Liu CG, et al (2007) CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. Proc Natl Acad Sci U S A 104: 2750–2755.
[62]
Garzon R, Garofalo M, Martelli MP, Briesewitz R, Wang L, et al. (2008) Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin. Proc Natl Acad Sci U S A 105: 3945–3950.
[63]
Krichevsky AM, Gabriely G (2009) miR-21: a small multi-faceted RNA. J Cell Mol Med 13: 39–53.
[64]
Si ML, Zhu S, Wu H, Lu Z, Wu F, et al. (2007) miR-21-mediated tumor growth. Oncogene 26: 2799–2803.
[65]
Qi L, Bart J, Tan LP, Platteel I, Sluis T, et al. (2009) Expression of miR-21 and its targets (PTEN, PDCD4, TM1) in flat epithelial atypia of the breast in relation to ductal carcinoma in situ and invasive carcinoma. BMC Cancer 9: 163.
[66]
Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, et al. (2008) Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem 283: 1026–1033.
[67]
Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH, et al. (2008) MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 27: 2128–2136.
[68]
Chen CZ, Lodish HF (2005) MicroRNAs as regulators of mammalian hematopoiesis. Semin Immunol 17: 155–165.
[69]
Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M, et al. (2007) Epigenetic silencing of the myelopoiesis regulator microRNA-223 by the AML1/ETO oncoprotein. Cancer Cell 12: 457–466.
[70]
Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, et al. (2008) Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature 451: 1125–1129.
[71]
Felli N, Pedini F, Romania P, Biffoni M, Morsilli O, et al. (2009) MicroRNA 223-dependent expression of LMO2 regulates normal erythropoiesis. Haematologica 94: 479–486.
[72]
Pulikkan JA, Dengler V, Peramangalam PS, Peer Zada AA, Muller-Tidow C, et al. (2010) Cell-cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia. Blood 115: 1768–1778.
[73]
Eyholzer M, Schmid S, Schardt JA, Haefliger S, Mueller BU, et al. (2010) Complexity of miR-223 regulation by CEBPA in human AML. Leuk Res 34: 672–676.
[74]
Radomska HS, Basseres DS, Zheng R, Zhang P, Dayaram T, et al. (2006) Block of C/EBP alpha function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations. J Exp Med 203: 371–381.
[75]
Boominathan L (2010) The tumor suppressors p53, p63, and p73 are regulators of microRNA processing complex. PLoS One 5: e10615.
[76]
Shin VY, Jin H, Ng EK, Cheng AS, Chong WW, et al. (2011) NF-kappaB targets miR-16 and miR-21 in gastric cancer: involvement of prostaglandin E receptors. Carcinogenesis 32: 240–245.
[77]
Zhang X, Chen X, Lin J, Lwin T, Wright G, et al. (2011) Myc represses miR-15a/miR-16–1 expression through recruitment of HDAC3 in mantle cell and other non-Hodgkin B-cell lymphomas. Oncogene.
[78]
Li L, Piloto O, Kim KT, Ye Z, Nguyen HB, et al. (2007) FLT3/ITD expression increases expansion, survival and entry into cell cycle of human haematopoietic stem/progenitor cells. Br J Haematol 137: 64–75.
[79]
Thomas M, Lange-Grunweller K, Weirauch U, Gutsch D, Aigner A, et al. (2011) The proto-oncogene Pim-1 is a target of miR-33a. Oncogene.
[80]
Chu SH, Small D (2009) Mechanisms of resistance to FLT3 inhibitors. Drug Resist Updat 12: 8–16.
[81]
Weisberg E, Barrett R, Liu Q, Stone R, Gray N, et al. (2009) FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist Updat 12: 81–89.
