Leukemia (2011) 25, 655–662 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE The transcription factor PlagL2 activates Mpl transcription and signaling in hematopoietic progenitor and leukemia cells SF Landrette1,3,4, D Madera1,3,FHe2 and LH Castilla1 1Program in Gene Function and Expression, Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA and 2Department of Microbiology and Physiologic Systems, University of Massachusetts Medical School, Worcester, MA, USA Cytokine signaling pathways are frequent targets of oncogenic factors, Plag1 and PlagL2, have similar capacity mutations in acute myeloid leukemia (AML), promoting to induce proliferation of HPs and cooperate with Cbfb-SMMHC proliferation and survival. We have previously shown that the in AML development.9,10 The PLAG factors bind to the transcription factor PLAGL2 promotes proliferation and cooperates with the leukemia fusion protein Cbfb-SMMHC in GRGGC(N)6À8RGGK consensus site in regulatory regions of AML development. Here, we show that PLAGL2 upregulates target genes to activate transcription, such as the promoter 3 of expression of the thrombopoietin receptor Mpl, using two the insulin-like growth factor 2 gene.11 The PLAG oncogenic consensus sites in its proximal promoter. We also show that activity has also been reported in chronic lymphocytic Mpl overexpression efficiently cooperates with Cbfb-SMMHC leukemia, breast cancer and salivary gland tumors.12–14 How- in development of leukemia in mice. Finally, we demonstrate ever, little is known on how PLAG induces transformation in that PlagL2-expressing leukemic cells show hyper-activation of Jak2 and downstream STAT5, Akt and Erk1/2 pathways in HPs and leukemia blasts. response to Thpo ligand. These results show that PlagL2 In this study, we use gene-expression profile analysis of HPs expression activates expression of Mpl in hematopoietic and leukemic cells expressing PLAGL2 to identify genes that are progenitors, and that upregulation of wild-type Mpl provides consistently deregulated by PLAGL2. We identify the thrombo- an oncogenic signal in cooperation with CBFb-SMMHC in mice. poietin receptor Mpl as a downstream target, using gene profile Leukemia (2011) 25, 655–662; doi:10.1038/leu.2010.301; analysis, and validated its expression levels using quantitative published online 25 January 2011 Keywords: acute myeloid leukemia; PlagL2; Mpl; Cbfb-MYH11; reverse transcription-PCR and flow cytometry. Furthermore, we gene regulation; transcription factors. identify two PLAG-binding sites conserved in mammals in Mpl proximal promoter using luciferase reporter and electro- phoretic mobility shift assays. We determined that Mpl is a key downstream mediator of PLAGL2 leukemogenesis as overexpression of wild-type Mpl efficiently cooperates with Introduction CBFb-SMMHC in leukemia development using transplantation assays. The leukemic cells expressing PLAGL2 exhibit sensitivity Acute myeloid leukemia (AML) results from the accumulation to Thpo ligand as evidenced by increased phosphorylation of of genetic alterations in hematopoietic progenitors (HPs) that Jak2, Erk1/2, Akt and Stat5. Together, these results demonstrate alter survival and differentiation programs. The study of these that PLAGL2 regulates Mpl expression, and that upregulation of alterations is key for understanding the mechanism of leukemia wild-type Mpl cooperates with CBFB-MYH11 in leukemia development. The chromosome 16 inversion inv(16)(p13;q22), development in mice. called inv16, is a frequent mutation found in AML cases, which creates the leukemia fusion gene CBFB-MYH11.1 The Cbfb- SMMHC protein encoded by the fusion gene induces accumula- Materials and methods tion of preleukemic myeloid progenitors unable to differentiate, which transform into full-blown leukemia in synergy with Microarray analyses mutations that promote proliferation and survival.2,3 Sample preparation. For infected bone marrow (BM)-HP A fraction of inv16-AML samples present activating mutations cells, 129SvEv Cbfb þ /56M;Mx1Cre mice were treated with in genes encoding components of the receptor tyrosine kinase 150 mg/kg 5-fluorouracil, and BM cells were harvested 5 days signaling pathways, including KIT and FLT3, and the their later. Cells were subjected to red-blood-cell lysis solution downstream GTPases NRAS and KRAS.4–8 These mutations (Purgene, Gentra Systems, Minneapolis, MN, USA), and spin- produce ligand-independent constitutively active signaling that infected with two rounds of retrovirus (MIG or MIG-PLAGL2) as enhances the expansion and survival of leukemic blasts. We previously described.10 The green-fluorescent protein (GFP)- have previously shown that the zinc finger PLAG transcription positive cells were sorted by fluorescence-activated cell sorting and total RNA was immediately isolated (samples wild-type Correspondence: Professor LH Castilla, Program in Gene Function and (wt)-MIG1, wt-MIG2, wt-P1 and wt-P2). The mouse AML cells Expression, University of Massachusetts Medical School, 364 Plantation were generated using Cbfb-MYH11 conditional knock-in mice Street, Worcester, MA 01605, USA. as previously described.3 AML cells were isolated from spleen of E-mail: [email protected] 3 three independent leukemic mice expressing MIG-PLAGL2 These authors contributed equally to this work. (samples AML-P1, AML-P2 and AML-P3) and from three 4Current address: Yale School of Medicine, 295 Congress Avenue, New Haven, CT 06519, USA. independent leukemic mice with low Plagl2 levels (AML-x1, Received 27 April 2010; revised 29 September 2010; accepted 15 AML-x2, AML-x3). In all cases, RNA was extracted with Trizol October 2010; published online 25 January 2011 (Invitrogen, Carlbad, CA, USA) and cleaned with RNA-Easy PlagL2 directly activates Mpl receptor SF Landrette et al 656 columns (Qiagen, Valencia, CA, USA). Complementary DNA manufacturer’s protocol. Retrovirus supernatants were taken at was synthesized using the Superscript II (Invitrogen), and 48, 56 and 72 h and titered in by GFP-fluorescence-activated Biotin-labeled cRNA was subsequently synthesized using cell sorting analysis. RNA-transcript labeling kit (Affymetrix, Santa Clara, CA, USA). Labeled cRNA (10 mg) was fragmented and hybridized to Affymetrix Mouse Genome 430 2.0 Chip Arrays (Affymetrix). Bone marrow transduction and transplantation Endogenous Cbfb-MYH11 expression was induced in Cbfb56M/ þ Mx1Cre conditional knock-in mice3 using the Mx1Cre trans- Data analysis genic system.15 Briefly, mice were injected with three doses To identify transcripts that were differentially expressed in every other day of polyinosinic-polycytidylic acid (Sigma, MIG-PLAGL2, AML-PLAGL2 and AML samples, we used the St Louis, MO, USA) at 3- to 5-weeks of age, treated with GeneSpring (version 7.3; http://www.silicongenetics.com) and 150 mg/kg 5-fluorouracil, and BM cells were harvested 5 days GENECLUSTER (version 2.0; http://www-genome.wi.mit.edu) later. Efficiency of Cre-mediated Cbfb-MYH11 induction was software packages. The expression values of each probe set in consistently above 90%.3 The BM progenitor cells were spin- each of the MIG-PLAGL2, AML-PLAGL2 and AML samples infected twice with retrovirus supernatant, and 5 Â 105–1 Â 106 were normalized to that of the MIG control number one or BM cells transplanted by intravenous injection (i.v.) into 4- to number two samples. For a given transcript to be considered as 6-week-old sublethally irradiated (650 rads) 129SvEv wild-type differentially expressed in MIG-PLAGL2, AML-PLAGL2 and recipient mice. Mice were under daily observation for early AML samples, it had to meet the following three criteria. First, signs of leukemia. These signs included limited motility, pale the signal values in these samples and the MIG control sample paws and dehydration. At first signs of illness, peripheral blood had to exhibit a relative difference of at least a factor of 2 and an was analyzed for the presence of immature cells. Fluorescence- absolute difference of 100 U. Second, changes of comparable activated cell sorting analysis of peripheral blood was performed magnitude had to be reproduced in two out two independent by using antibodies to cell-surface markers Gr-1, CD11b, B220, experiments for the MIG-PLAG2 sample or three out three CD3, Ter119 and c-kit (BD Biosciences, San Diego, CA, USA). independent experiments for the AML-PLAGL2 and AML For flow cytometry analysis of Mpl receptor expression, a samples when compared with MIG controls. Third, when polyclonal rabbit anti-Mpl extracellular domain antibody statistical group comparisons (Welch’s approximate t-test) were (provided by Harvey Lodish, Whitehead Institute for Biomedical applied to the raw signal values of the MIG control samples and Research, Cambridge, MA, USA) and a PE-anti-rabbit secondary the MIG-PLAGL2, AML-PLAGL2 or AML samples, it yielded antibody were used. For secondary transplantations, leukemic a significance value of Pp0.05. Transcripts differentially cells were harvested from the BM or spleen of affected mice in expressed in MIG-PLAGL2, AML-PLAGL2 or AML samples RPMI 1640 and 20% fetal bovine serum (both from Invitrogen), were subjected to hierarchical clustering analysis using the and 1 Â 106 single-cell suspension aliquots were transplanted GeneSpring (version 7.3) software package. For this analysis, the i.v. into sub-lethally irradiated 4- to 6-week-old 129SvEv signal value of each transcript in each of the samples was recipients.