Abstract
It is now conceivable that leukemogenesis requires two types of mutations, class I and class II mutations. We previously established a mouse bone marrow-derived HF6, an IL-3-dependent cell line, that was immortalized by a class II mutation MLL/SEPT6 and can be fully transformed by class I mutations such as FLT3 mutants. To understand the molecular mechanism of leukemogenesis, particularly progression of myelodysplastic syndrome (MDS) to acute leukemia, we made cDNA libraries from the samples of patients and screened them by expression-cloning to detect class I mutations that render HF6 cells factor-independent. We identified RasGRP4, an activator of Ras, as a candidate for class I mutation from three of six patients (MDS/MPD = 1, MDS-RA = 1, MDS/AML = 2, CMMoL/AML = 1 and AML-M2 = 1). To investigate the potential roles of RasGRP4 in leukemogenesis, we tested its in vivo effect in a mouse bone marrow transplantation (BMT) model. C57BL/6J mice transplanted with RasGRP4-transduced primary bone marrow cells died of T cell leukemia, myeloid leukemia, or myeloid leukemia with T cell leukemia. To further examine if the combination of class I and class II mutations accelerated leukemic transformation, we performed a mouse BMT model in which both AML1 mutant (S291fsX300) and RasGRP4 were transduced into bone marrow cells. The double transduction led to early onset of T cell leukemia but not of AML in the transplanted mice when compared to transduction of RasGRP4 alone. Thus, we have identified RasGRP4 as a gene potentially involved in leukemogenesis and suggest that RasGRP4 cooperates with AML1 mutations in T cell leukemogenesis as a class I mutation.
Similar content being viewed by others
References
Beghini A, Peterlongo P, Ripamonti CB, Larizza L, Cairoli R, Morra E, et al. C-kit mutations in core binding factor leukemias. Blood. 2000;95:726–7.
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. Proc Natl Acad Sci USA. 2005;102:1104–9. doi:10.1073/pnas.0408831102.
Shimada A, Taki T, Tabuchi K, Tawa A, Horibe K, Tsuchida M, et al. KIT mutations, and not FLT3 internal tandem duplication, are strongly associated with a poor prognosis in pediatric acute myeloid leukemia with t(8;21): a study of the Japanese Childhood AML Cooperative Study Group. Blood. 2006;107:1806–9. doi:10.1182/blood-2005-08-3408.
Yamashita N, Osato M, Huang L, Yanagida M, Kogan SC, Iwasaki M, et al. Haploinsufficiency of Runx1/AML1 promotes myeloid features and leukemogenesis in BXH2 mice. Br J Haematol. 2005;131:495–507. doi:10.1111/j.1365-2141.2005.05793.x.
Care RS, Valk PJ, Goodeve AC, Abu-Duhier FM, Geertsma-Kleinekoort WM, Wilson GA, et al. Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol. 2003;121:775–7. doi:10.1046/j.1365-2141.2003.04362.x.
Boissel N, Leroy H, Brethon B, Philippe N, de Botton S, Auvrignon A, et al. Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia. 2006;20:965–70. doi:10.1038/sj.leu.2404188.
Beghini A, Ripamonti CB, Cairoli R, Cazzaniga G, Colapietro P, Elice F, et al. KIT activating mutations: incidence in adult and pediatric acute myeloid leukemia, and identification of an internal tandem duplication. Haematologica. 2004;89:920–5.
Christiansen DH, Andersen MK, Desta F, Pedersen-Bjergaard J. Mutations of genes in the receptor tyrosine kinase (RTK)/RAS-BRAF signal transduction pathway in therapy-related myelodysplasia and acute myeloid leukemia. Leukemia. 2005;19:2232–40. doi:10.1038/sj.leu.2404009.
Niimi H, Harada H, Harada Y, Ding Y, Imagawa J, Inaba T, et al. Hyperactivation of the RAS signaling pathway in myelodysplastic syndrome with AML1/RUNX1 point mutations. Leukemia. 2006;20:635–44. doi:10.1038/sj.leu.2404136.
Matsuno N, Osato M, Yamashita N, Yanagida M, Nanri T, Fukushima T, et al. Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype. Leukemia. 2003;17:2492–9. doi:10.1038/sj.leu.2403160.
Roumier C, Eclache V, Imbert M, Davi F, MacIntyre E, Garand R, et al. M0 AML, clinical and biologic features of the disease, including AML1 gene mutations: a report of 59 cases by the Groupe Français d’Hématologie Cellulaire (GFHC) and the Groupe Français de Cytogénétique Hématologique (GFCH). Blood. 2003;101:1277–83. doi:10.1182/blood-2002-05-1474.
Callens C, Chevret S, Cayuela JM, Cassinat B, Raffoux E, de Botton S, et al. Prognostic implication of FLT3 and Ras gene mutations in patients with acute promyelocytic leukemia (APL): a retrospective study from the European APL Group. Leukemia. 2005;19:1153–60. doi:10.1038/sj.leu.2403790.
Gale RE, Hills R, Pizzey AR, Kottaridis PD, Swirsky D, Gilkes AF, et al. Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia. Blood. 2005;106:3768–76. doi:10.1182/blood-2005-04-1746.
Arrigoni P, Beretta C, Silvestri D, Rossi V, Rizzari C, Valsecchi MG, et al. FLT3 internal tandem duplication in childhood acute myeloid leukaemia: association with hyperleucocytosis in acute promyelocytic leukaemia. Br J Haematol. 2003;120:89–92. doi:10.1046/j.1365-2141.2003.04032.x.
Noguera NI, Breccia M, Divona M, Diverio D, Costa V, De Santis S, et al. Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol. Leukemia. 2002;16:2185–9. doi:10.1038/sj.leu.2402723.
Kainz B, Heintel D, Marculescu R, Schwarzinger I, Sperr W, Le T, et al. Variable prognostic value of FLT3 internal tandem duplications in patients with de novo AML and a normal karyotype, t(15;17), t(8;21) or inv(16). Hematol J. 2002;3:283–9. doi:10.1038/sj.thj.6200196.
Taketani T, Taki T, Sugita K, Furuichi Y, Ishii E, Hanada R, et al. FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood. 2004;103:1085–8. doi:10.1182/blood-2003-02-0418.
Liang DC, Shih LY, Fu JF, Li HY, Wang HI, Hung IJ, et al. K-Ras mutations and N-Ras mutations in childhood acute leukemias with or without mixed-lineage leukemia gene rearrangements. Cancer. 2006;106:950–6. doi:10.1002/cncr.21687.
Gale RE, Green C, Allen C, Mead AJ, Burnett AK, Hills RK, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia. Blood. 2008;111:2776–84. doi:10.1182/blood-2007-08-109090.
Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S, et al. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood. 1999;93:3074–80.
Stirewalt DL, Kopecky KJ, Meshinchi S, Appelbaum FR, Slovak ML, Willman CL, et al. FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia. Blood. 2001;97:3589–95. doi:10.1182/blood.V97.11.3589.
Kelly LM, Kutok JL, Williams IR, Boulton CL, Amaral SM, Curley DP, et al. PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model. Proc Natl Acad Sci USA. 2002;99:8283–8. doi:10.1073/pnas.122233699.
Warner JK, Wang JC, Takenaka K, Doulatov S, McKenzie JL, Harrington L, et al. Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells. Leukemia. 2005;19:1794–805. doi:10.1038/sj.leu.2403917.
Gilliland DG, Griffin JD. Role of FLT3 in leukemia. Curr Opin Hematol. 2002;9:274–81. doi:10.1097/00062752-200207000-00003.
Schessl C, Rawat VP, Cusan M, Deshpande A, Kohl TM, Rosten PM, et al. The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice. J Clin Invest. 2005;115:2159–68. doi:10.1172/JCI24225.
Cuenco GM, Ren R. Cooperation of BCR-ABL and AML1/MDS1/EVI1 in blocking myeloid differentiation and rapid induction of an acute myelogenous leukemia. Oncogene. 2001;20:8236–48. doi:10.1038/sj.onc.1205095.
Ono R, Nakajima H, Ozaki K, Kumagai H, Kawashima T, Taki T, et al. Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis. J Clin Invest. 2005;115:919–29.
Chan IT, Kutok JL, Williams IR, Cohen S, Moore S, Shigematsu H, et al. Oncogenic K-ras cooperates with PML-RAR alpha to induce an acute promyelocytic leukemia-like disease. Blood. 2006;108:1708–15. doi:10.1182/blood-2006-04-015040.
Reuther GW, Lambert QT, Rebhun JF, Caligiuri MA, Quilliam LA, Der CJ. RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia. J Biol Chem. 2002;277:30508–14. doi:10.1074/jbc.M111330200.
Yang Y, Li L, Wong GW, Krilis SA, Madhusudhan MS, Sali A, et al. RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function. J Biol Chem. 2002;277:25756–74. doi:10.1074/jbc.M202575200.
Kitamura T, Onishi M, Kinoshita S, Shibuya A, Miyajima A, Nolan GP. Efficient screening of retroviral cDNA expression libraries. Proc Natl Acad Sci USA. 1995;92:9146–50. doi:10.1073/pnas.92.20.9146.
Kitamura T, Koshino Y, Shibata F, Oki T, Nakajima H, Nosaka T, et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Exp Hematol. 2003;31:1007–14.
Harada H, Harada Y, Niimi H, Kyo T, Kimura A, Inaba T. High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia. Blood. 2004;103:2316–24. doi:10.1182/blood-2003-09-3074.
Izawa K, Kitaura J, Yamanishi Y, Matsuoka T, Oki T, Shibata F, et al. Functional analysis of activating receptor LMIR4 as a counterpart of inhibitory receptor LMIR3. J Biol Chem. 2007;282:17997–8008. doi:10.1074/jbc.M701100200.
Watanabe-Okochi N, Kitaura J, Ono R, Harada H, Harada Y, Komeno Y, et al. AML1 mutations induced MDS and MDS/AML in a mouse BMT model. Blood. 2008;111:4297–308. doi:10.1182/blood-2007-01-068346.
Reuss-Borst MA, Bühring HJ, Schmidt H, Müller CA. AML: immunophenotypic heterogeneity and prognostic significance of c-kit expression. Leukemia. 1994;8:258–63.
Chinen Y, Taki T, Nishida K, Shimizu D, Okuda T, Yoshida N, et al. Identification of the novel AML1 fusion partner gene, LAF4, a fusion partner of MLL, in childhood T cell acute lymphoblastic leukemia with t(2;21)(q11;q22) by bubble PCR method for cDNA. Oncogene. 2008;27:2249–56. doi:10.1038/sj.onc.1210857.
Mikhail FM, Coignet L, Hatem N, Mourad ZI, Farawela HM, El Kaffash DM, et al. A novel gene, FGA7, is fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a patient with T cell acute lymphoblastic leukemia. Genes Chromosomes Cancer. 2004;39:110–8. doi:10.1002/gcc.10302.
Kitamura T. New experimental approaches in retrovirus-mediated expression screening. Int J Hematol. 1998;67:351–9. doi:10.1016/S0925-5710(98)00025-5.
Acknowledgments
We thank Fumi Shibata for technical assistance and Dr. Yusuke Nakamura for the SNPs information of RasGRP4. This work was supported by a grant-in-aid for Cancer Research supported by the Ministry of Health, Labor and Welfare and a grant from the Vehicle Racing Commemorative Foundation.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Watanabe-Okochi, N., Oki, T., Komeno, Y. et al. Possible involvement of RasGRP4 in leukemogenesis. Int J Hematol 89, 470–481 (2009). https://doi.org/10.1007/s12185-009-0299-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12185-009-0299-0