OPEN Leukemia (2017) 31, 2543–2551 www.nature.com/leu ORIGINAL ARTICLE Dysfunction of the WT1-MEG3 signaling promotes AML leukemogenesis via p53-dependent and -independent pathways YLyu1,2,7, J Lou1,3,7, Y Yang1,2,7, J Feng1,7, Y Hao1, S Huang1, L Yin1,2,JXu1, D Huang1,2,BMa1,3, D Zou1, Y Wang1,2, Y Zhang1, B Zhang3, P Chen4,KYu5, EW-F Lam6, X Wang1, Q Liu1,JYan1,2 and B Jin1 Long non-coding RNAs (lncRNAs) play a pivotal role in tumorigenesis, exemplified by the recent finding that lncRNA maternally expressed gene 3 (MEG3) inhibits tumor growth in a p53-dependent manner. Acute myeloid leukemia (AML) is the most common malignant myeloid disorder in adults, and TP53 mutations or loss are frequently detected in patients with therapy-related AML or AML with complex karyotype. Here, we reveal that MEG3 is significantly downregulated in AML and suppresses leukemogenesis not only in a p53-dependent, but also a p53-independent manner. In addition, MEG3 is proven to be transcriptionally activated by Wilms’ tumor 1 (WT1), dysregulation of which by epigenetic silencing or mutations is causally involved in AML. Therefore MEG3 is identified as a novel target of the WT1 molecule. Ten–eleven translocation-2 (TET2) mutations frequently occur in AML and significantly promote leukemogenesis of this disorder. In our study, TET2, acting as a cofactor of WT1, increases MEG3 expression. Taken together, our work demonstrates that TET2 dysregulated WT1-MEG3 axis significantly promotes AML leukemogenesis, paving a new avenue for diagnosis and treatment of AML patients. Leukemia (2017) 31, 2543–2551; doi:10.1038/leu.2017.116 INTRODUCTION the regulation of the RB pathway and thus of cell proliferation,11,12 Acute myeloid leukemia (AML) is the most common malignant implying a possible role in a p53-independent pathway. The RB myeloid disorder in adults, which is a heterogeneous clonal protein can repress gene transcription by directly binding to the disorder of hematopoietic progenitor cells, with diverse biological, transactivation domain of E2F in a complex on the promoters of 13 phenotypic and prognostic behaviors, and strikingly different the E2F target genes including DNMT3A. Accordingly, RB is outcomes to standard therapy.1,2 In order to improve outcome in involved in transcriptional regulation of DNMT3A gene in cancer AML, current efforts in basic and clinical research focus on the cells,13 however, the detailed role of the RB-DNMT3A pathway genetic characterization of AML in the hopes of furthering our remains to be fully elucidated. understanding of AML pathogenesis and discovering new The Wilms’ tumor 1 (WT1) gene, located on chromosome 11p13, targeted therapies that will eventually lead to an increase in the encodes a transcriptional regulator that is capable of activating or cure rate.2,3 Long non-coding RNAs (lncRNAs), defined as repressing gene transcription.14,15 The precise role of WT1 in transcripts longer than 200 nucleotides,4 affect various cellular hematopoiesis and its contribution to leukemogenesis are open functions such as gene regulation, genomic imprinting, RNA for speculation. It is reported that WT1 mutations are associated maturation and translation.5,6 Moreover, multiple lines of evidence with an extremely poor outcome,15 and they can lead to link dysregulation of lncRNAs to diverse human diseases, progression of leukemia by conferring drug resistance.16,17 especially cancer. In the process of carcinogenesis, certain lncRNAs Dysregulation of the WT1 gene by epigenetic modifications or have been attributed to oncogenic and/or tumor suppressor mutations might promote leukemic cell proliferation and impair roles.5 differentiation.18,19 However, the role of WT1 in regulating cancer- Maternally expressed gene 3 (MEG3) is a myeloid-related related gene expression remains largely unknown, especially lncRNA that has been shown to act as tumor suppressor in solid whether WT1 controls MEG3 activity has yet to be explored. tumors.7,8 Previous data have demonstrated that MEG3 is capable The ten–eleven translocation (TET) family proteins TET1, TET2 of increasing the protein level of the tumor suppressor p53 and and TET3 constitute a novel family of dioxygenases, whose enhancing p53 binding to its target promoters.9 In consequence, it functions are to demethylate DNA sequence by converting is very evident that MEG3 can inhibit tumorigenesis through a 5-methylcytosine to 5-hydroxymethylcytosine.20 Pathologically, p53-dependent manner. However, TP53 mutations can result in TET2 is frequently mutated in hematopoietic malignancies of the loss of wild-type tumor-suppressing p53 function in diverse types myeloid lineage, particularly in AML.21 Most recently, WT1 is found of human cancer, including AML.10 MEG3 has been implicated in to physically interact with TET2 and recruit it to the target genes of 1Department of Hematology, the Second Affiliated Hospital, Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, Dalian, China; 2Department of Hematology, the Second Affiliated Hospital, Institute of Hematopoeitic Stem Cell Transplantation of Dalian Medical University, Liaoning Hematopoeitic Stem Cell Transplantation Medical Center, Dalian Key Laboratory of Hematology, Dalian Medical University, Dalian, China; 3Department of Neurosurgery, the Second Affiliated Hospital of Dalian Medical University, Dalian, China; 4Department of Obstetrics and Gynecology, the Second Xiangya Hospital, Central South University, Changsha, China; 5Department of Cellular Biology and Anatomy, Augusta University, Augusta, GA, USA and 6Department of Surgery and Cancer, Imperial College London, London, UK. Correspondence: Professor B Jin or Professor J Yan or Professor Q Liu or Dr X Wang, Department of Hematology, the Second Affiliated Hospital, Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, No.9 West Section, Lvshun South Road, Dalian 116023, China. E-mail: [email protected] or [email protected] or [email protected] or [email protected] 7These authors contributed equally to this work. Received 26 August 2016; revised 16 March 2017; accepted 4 April 2017; accepted article preview online 12 April 2017; advance online publication, 2 May 2017 WT1–TET2 complex regulates LncRNA MEG3 in AML Y Lyu et al 2544 WT1,22 suggesting that TET2 may be involved in the transcrip- Statistical analysis tional activity of WT1 in AML. Student’s t-test (two-tailed), t-test with Welch's correction, Log-rank In this study, we demonstrate that inactivation of MEG3 (Mantel–Cox) test, F-test were performed to analyze the data using promotes AML leukemogenesis in a p53-dependent as well as a GraphPad Prism 6.0 software (GraphPad software Inc., San Diego, CA, USA). fi P-values o0.05 were considered statistically significant. *Po0.05; p53-independent mode. Further analyses show that WT1 speci - o o cally binds to the MEG3 promoter and activates its transcription, **P 0.01; ***P 0.001. and thereby, inducing a reduction of its downstream molecule MDM2. We further investigate the possible association between RESULTS TET2 and WT1 in AML, and the results suggest that TET2 acts as a LncRNA MEG3 is downregulated in AML cofactor of WT1 to promote MEG3 transcription. Given the To gain insights into the patterns of activation of MEG3 gene in importance of the WT1-MEG3 axis in suppressing tumor growth, fi the AML pathogenesis, we examined its expression in normal our ndings suggest that targeting this axis may represent a novel CD34+ bone marrow cells and AML patient samples with different approach for effective AML treatment. WT1 or TET2 mutation status. Given some variations were usually not regarded as true missense mutations (including P29R, I1762V, V218M, L1721W and H1778R) in TET2,23 only nonsense (4/42, 9.5%) and frameshift mutations (9/42, 21.4%) were used in our MATERIALS AND METHODS analysis. WT1 mutations (frameshift) were detected in two patients More detailed information on materials and methods can be found in the (2/42, 4.8%; Supplementary Tables 1 and 2). Real-time quantitative Supplementary Information. PCR results indicated that MEG3 was robustly expressed in bone marrow CD34+ cells, significantly downregulated in all AML samples, particularly in the WT1-orTET2-mutant AML subtypes Patient and tissue samples (Figures 1a and b). Forty-two AML patient samples were analyzed at the time of diagnosis in For subsequent cell model experiments, we also investigated fi – – this study. AML patients were classi ed according to French American the genetic backgrounds (Supplementary Tables 1 and 2) and p53 British. All patients gave their written informed consent. The study has levels (Supplementary Figure 1A) of eight representative cell lines been approved by the Ethics Committee of the Institute. Mononuclear cells derived from AML (K562, TF-1, MOLM-13, U937, NB4, Kasumi-1, were isolated by density gradient centrifugation using Lymphoprep, and cryopreserved. In addition, 15 potential donors for allogeneic bone marrow KG-1 and HL-60) and the correlations between WT1, TET2 mutation transplantation were used as normal controls. Highly enriched human status/expression levels and MEG3 inactivation in these cells CD34+ cells (490%) were derived from bone marrow mononuclear cells (Supplementary Figure 1B). As expected, MEG3 displayed negli- using MiniMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) following gible expression in the WT1-mutant AML cell lines (U937) relative the manufacturer’s instructions. Confirmation of bone marrow-derived to WT1- and TET2-wild-type