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188 between hemizygous loss of chromosome material and downregulation of targeted miRNAs.5 The pathogenically relevant target gene(s) at der(9q) remains to be identified.
Acknowledgements This study was supported by a grant from the Department of Defense (CM050018) to RCTA. Ricardo CT Aguiar is a Scholar of the American Society of Hematology.
A Chaubey2, S Karanti1, D Rai1, T Oh1, SG Adhvaryu2 and RCT Aguiar1 1 Division of Hematology and Medical Oncology, Department of Medicine, Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA and 2 Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA E-mail:
[email protected]
References 1 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297. 2 Esquela-Kerscher A, Slack FJ. OncomirsFmicroRNAs with a role in cancer. Nat Rev Cancer 2006; 6: 259–269.
3 He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S et al. A microRNA polycistron as a potential human oncogene. Nature 2005; 435: 828–833. 4 Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002; 99: 15524–15529. 5 Fulci V, Chiaretti S, Goldoni M, Azzalin G, Carucci N, Tavolaro S et al. Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia. Blood 2007; 109: 4944–4951. 6 Fourouclas N, Campbell PJ, Bench AJ, Swanton S, Baxter EJ, Huntly BJ et al. Size matters: the prognostic implications of large and small deletions of the derivative 9 chromosome in chronic myeloid leukemia. Haematologica 2006; 91: 952–955. 7 Kreil S, Pfirrmann M, Haferlach C, Waghorn K, Chase A, Hehlmann R et al. Heterogeneous prognostic impact of derivative chromosome 9 deletions in chronic myelogenous leukemia. Blood 2007; 110: 1283–1290. 8 Rai D, Karanti S, Jung I, Dahia PL, Aguiar RC. Coordinated expression of microRNA-155 and predicted target genes in diffuse large B-cell lymphoma. Cancer Genet Cytogenet 2008; 181: 8–15. 9 Reid AG, Tarpey PS, Nacheva EP. High-resolution analysis of acquired genomic imbalances in bone marrow samples from chronic myeloid leukemia patients by use of multiple short DNA probes. Genes Chromosomes Cancer 2003; 37: 282–290. 10 Wilkinson K, Velloso ER, Lopes LF, Lee C, Aster JC, Shipp MA et al. Cloning of the t(1;5)(q23;q33) in a myeloproliferative disorder associated with eosinophilia: involvement of PDGFRB and response to imatinib. Blood 2003; 102: 4187–4190.
Transient depletion of RUNX1/RUNX1T1 by RNA interference delays tumour formation in vivo
Leukemia (2009) 23, 188–190; doi:10.1038/leu.2008.157; published online 12 June 2008
Chromosomal translocation t(8;21)(q22;q22) occurs in about 10% of all cases of acute myeloid leukaemia (AML) and is one of the most frequent chromosomal abnormalities found in AML.1 This translocation generates the RUNX1/RUNX1T1 (AML1/ MTG8, AML1/ETO) fusion gene, which by itself is not sufficient for full leukaemic transformation, but which supports human haematopoietic stem/progenitor cell self-renewal in vivo as well as leukaemic proliferation and clonogenicity ex vivo.2–4 Generation of RUNX1/RUNX1T1 may not only be an initiating event in leukaemogenesis, but might also become a leukaemiaspecific target for therapeutic approaches. However, its role in leukaemic persistence in vivo and, in particular, its significance for leukaemic stem cells has not been established yet. One prerequisite for studying possible functions of RUNX1/ RUNX1T1 in maintaining leukaemia in vivo and the development of therapies targeting this genetic lesion is the availability of suitable animal model systems. Several transgenic and knockin mouse models have been developed to study RUNX1/ RUNX1T1-driven leukaemogenesis in murine haematopoietic cells.5 However, in vivo studies of RUNX1/RUNX1T1 in the human leukaemic background are currently limited due to a lack of suitable xenotransplantation models, as t(8;21)-positive leukaemic cells including the established cell lines do not or only rarely engraft in Nod/Scid mouse strains.6 The Rag2/gc/ mouse strain, which lacks B, T and natural killer cells represents an interesting alternative to Scid-based Leukemia
mouse strains. For instance, intrahepatic injection of CD34 þ human cord blood cells into newborn BALB/c Rag2/gc/ mice leads to the development of an adaptive immune system.7 We tested this immunodeficient mouse strain in combination with the t(8;21)-positive AML cell line Kasumi-1 to establish a xenotransplantation model for t(8;21)-associated AML. Sublethally irradiated newborn mice received an intraperitoneal injection of 106 Kasumi-1 cells. Within 53–55 days, transplanted mice developed swollen abdomen or showed other signs of tumour formation. Postmortem examination revealed mainly solid tumours of 1 cm–1.5 cm in diameter (Figure 1a) located intraperitoneally. Tumour histology and flow cytometry analyses revealed that the tumours consisted almost exclusively of human myeloid CD45 þ , CD34 þ and CD33 þ cells, which is concordant with the immunophenotype of Kasumi-1 cells (Figures 1b and c and data not shown). Notably, such extramedullary myeloid tumours (granulocytic sarcomas and chloromas) are found in some 20% of all t(8;21) AML cases.8 In less than half of the animals, we observed infiltration of the spleen by leukaemic cells, but neither bone marrow nor liver infiltration (Figure 1c). Consistent with this, RUNX1/RUNX1T1 protein was strongly expressed in tumours, occasionally and weakly in spleen and never in liver (Figure 1d). We recently analysed the significance of RUNX1/RUNX1T1 for leukaemic proliferation and clonogenicity using short interfering RNAs (siRNAs). A single siRNA treatment caused a transient reduction in fusion protein levels lasting 5–7 days. siRNA-mediated RUNX1/RUNX1T1 depletion restored myeloid differentiation capacity, inhibited proliferation and severely impaired leukaemic clonogenicity in vitro.3,4 However, these
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Figure 1 Analysis of Rag2/gc/ mice xenotransplanted with t(8;21) AML cells. Newborn mice were transplanted with t(8;21)positive Kasumi-1 cells. Animals had to be humanely killed within 50–70 days because of tumour burden. (a) Tumour excised from the abdominal cavity of an animal transplanted with 106 cells. (b) Tumour histology. The animal was transplanted with 2.5 105 cells pretreated with the mismatch control short interfering RNA (siRNA) siAGF6. The tumour was stained with hematoxylin/eosin. The original magnification was 20. (c) Flow cytometry analysis of tumour-, spleen- and bone marrow-derived cells. Examples for infiltrated (top right) and non-infiltrated spleens (bottom right) are shown. The red curves show the cells stained with a-human CD45 antibody, the blue curves show the isotype controls. (d) Immunoblot analysis for RUNX1/RUNX1T1 expression. Tissues are indicated at the top, detected proteins on the right and length marker size on the left. Mice 1 and 2 were transplanted animals; control indicates a non-transplanted control animal. Kasumi-1 cells served as positive control. RUNX1/RUNX1T1 was detected with an anti-AML1 antibody as described.4 Tubulin (TUB) served as a loading control. Note the RUNX1/RUNX1T1 signal in spleen lysate from animal 2 indicating weak leukaemic spleen infiltration.
experiments did not address the in vivo significance of this leukaemic fusion protein in human leukaemic cells. To examine the consequences of a transient RUNX1/RUNX1T1 depletion on leukaemic engraftment and tumour formation, we used the Rag2/gc/ transplantation system. We electroporated Kasumi-1 cells either in the absence of any siRNA (‘mock’), with siRNA targeting the RUNX1/RUNX1T1 fusion site (‘siAGF1’) or
Figure 2 Short interfering RNA (siRNA)-mediated RUNX1/RUNX1T1 suppression decreases leukaemic clonogenicity and extends median survival. Kasumi-1 cells were electroporated with the indicated siRNAs as described previously.4 Cells were examined for RUNX1/ RUNX1T1 protein levels and colony formation in vitro, and transplanted into newborn Rag2/gc/ mice. (a) Immunoblot for RUNX1/RUNX1T1 expression in siRNA-treated Kasumi-1 cells. siRNA treatments are indicated at the top, detected proteins on the right and length marker size on the left. RUNX1/RUNX1T1 and RUNX1 were detected with an anti-AML1 antibody as described.4 Tubulin (TUB) served as a loading control. (b) Colony formation of siRNA-treated Kasumi-1 cells. Colony formation assays were performed as described.4 CFC, colony forming cells. Error bars indicate standard deviations. (c) Survival curves of Rag2/gc/ mice transplanted with 106 siRNA-treated Kasumi-1 cells. Pretreatment with the RUNX1/ RUNX1T1 siRNA siAGF1 extended median survival significantly compared to mock or control siRNA siAGF6 pretreatment (Po0.02 according to log-rank test). In each treatment arm, at least 10 mice were transplanted. (d) Survival curves of Rag2/gc/ mice transplanted with 2.5 105 siRNA-treated Kasumi-1 cells. Pretreatment with the RUNX1/RUNX1T1 siRNA siAGF1 extended median survival significantly compared to control siRNA siAGF6 pretreatment (Po0.02 according to log-rank test). In each treatment arm, at least seven mice were transplanted.
with a mismatch control siRNA (‘siAGF6’) as described previously.3 siRNA-mediated RUNX1/RUNX1T1 suppression was examined by western blotting and functionally analysed Leukemia
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by colony formation assays.4 In comparison to mock and mismatch siRNA-treated cells, siAGF1-treated cells showed at least a threefold decrease in fusion protein and a sixfold decrease in colony forming cells in vitro (Figures 2a and b). One day after siRNA treatment, cells were either transplanted or stored in liquid nitrogen till transplantation. Transplantations were performed by intraperitoneally injecting 106 electroporated cells into sublethally irradiated newborn mice. Total group sizes were 12 animals for the mock group, 11 animals for the active siRNA group and 10 animals for the mismatch control siRNA group. Transplantation of mock- or mismatch siRNAtreated cells resulted in tumour formation in all transplanted animals with a median survival of 50 days (Figure 2c). In contrast, pretreatment with the active RUNX1/RUNX1T1 siRNA siAGF1 resulted in an extended median survival of 73 days (Po0.02). Notably, three animals of the active siRNA group examined 71 days post-transplantation showed no signs of tumour formation in histological analyses. In a second set of experiments, 2.5 105 cells were transplanted 8 h after siRNA electroporation into non-irradiated mice. In this experiment, groups of eight and seven animals were transplanted with active and mismatch siRNA-pretreated cells. This setting resulted in a median survival of 64 and 90 days for the mismatch siRNA and the active siRNA group, respectively. Again, the difference in median survival was statistically significant (Po0.02) suggesting that siRNA-mediated transient reduction of RUNX1/RUNX1T1 causes a substantial decrease in cancer-initiating cells. In summary, we show that already a transient siRNA-mediated depletion of RUNX1/RUNX1T1 causes a significant increase in median survival in a xenotransplantation model. These findings suggest that RUNX1/RUNX1T1 siRNAs compromises the engraftment and/or self-renewal capacities of t(8;21) leukaemiainitiating cells. Future studies will show whether RNAi-mediated RUNX1/RUNX1T1 suppression during and after leukaemic engraftment may stop or even reverse tumour formation, consequently paving the way for developing therapeutic approaches directly targeting this leukaemic fusion protein.
N Martinez Soria1, R Tussiwand2, P Ziegler2, MG Manz2 and O Heidenreich1,3
1 Department of Molecular Biology, Interfaculty Institute for Cell Biology, Eberhard Karls University Tuebingen, Tuebingen, Germany; 2 Hematopoiesis Laboratory, Institute for Research in Biomedicine, Bellinzona, Switzerland and 3 Molecular Paediatric Oncology Laboratory, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK E-mail:
[email protected]
References 1 Downing JR. The AML1-ETO chimaeric transcription factor in acute myeloid leukaemia: biology and clinical significance. Br J Haematol 1999; 106: 296–308. 2 Mulloy JC, Cammenga J, Berguido FJ, Wu K, Zhou P, Comenzo RL et al. Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element. Blood 2003; 102: 4369–4376. 3 Heidenreich O, Krauter J, Riehle H, Hadwiger P, John M, Heil G et al. AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells. Blood 2003; 101: 3157–3163. 4 Martinez N, Drescher B, Riehle H, Cullmann C, Vornlocher HP, Ganser A et al. The oncogenic fusion protein RUNX1-CBFA2T1 supports proliferation and inhibits senescence in t(8;21)-positive leukaemic cells. BMC Cancer 2004; 4: 44. 5 McCormack E, Bruserud O, Gjertsen BT. Review: genetic models of acute myeloid leukaemia. Oncogene, advance online publication 11 February 2008: DOI 10.1038/ onc.2008.1016. 6 Pearce DJ, Taussig D, Zibara K, Smith LL, Ridler CM, Preudhomme C et al. AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML. Blood 2006; 107: 1166–1173. 7 Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A et al. Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 2004; 304: 104–107. 8 Tallman MS, Hakimian D, Shaw JM, Lissner GS, Russell EJ, Variakojis D. Granulocytic sarcoma is associated with the 8;21 translocation in acute myeloid leukemia. J Clin Oncol 1993; 11: 690–697.
Prior treatment with the tyrosine kinase inhibitors dasatinib and nilotinib allows stem cell transplantation (SCT) in a less advanced disease phase and does not increase SCT Toxicity in patients with chronic myelogenous leukemia and philadelphia positive acute lymphoblastic leukemia
Leukemia (2009) 23, 190–194; doi:10.1038/leu.2008.160; published online 3 July 2008
Chronic myeloid leukemia (CML) is a clonal disorder associated with chromosomal translocation t(9;22), which produces the Philadelphia chromosome.1 The fusion gene encodes for the chimeric oncoprotein BCR-ABL, associated with deregulated constitutive tyrosine kinase (TK) activity, leading to leukemogenesis.1 Imatinib mesylate, the first potent selective inhibitor of BCR-ABL TK, has become the frontline therapy for newly diagnosed CML patients.1 A large phase III study (the IRIS study) showed that imatinib can induce complete cytogenetic response (CcyR) in 87% of recently diagnosed patients with CML in chronic phase (CP).2 Imatinib is also effective in advanced phase Leukemia
disease; however, in general, the response is short-lived.1 Imatinib has also been incorporated into therapeutic regimens in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph þ ALL). The introduction of imatinib markedly reduced the use of allogeneic hematopoietic stem cell transplantation (SCT) in CML in the first CP. SCT is currently reserved for patients in CP after the failure of imatinib or for patients in advanced phase disease. SCT remains the treatment of choice in patients with Ph þ ALL. Most studies have demonstrated that imatinib therapy before allogeneic SCT does not adversely affect the transplantation outcome and that imatinib can also be used successfully as a bridge to SCT in advanced disease.3 Resistance to imatinib is an increasingly recognized problem, and it is either primary or acquired. The most common cause for
