A Double Negative Loop Comprising ETV6/RUNX1 and MIR181A1 Contributes to Differentiation Block in t(12;21)-Positive Acute Lymphoblastic Leukemia

RESEARCH ARTICLE A Double Negative Loop Comprising ETV6/ RUNX1 and MIR181A1 Contributes to Differentiation Block in t(12;21)-Positive Acute Lymphoblastic Leukemia Yung-Li Yang1,2☯, Ching-Tzu Yen3☯, Chen-Hsueh Pai3, Hsuan-Yu Chen4, Sung-Liang Yu3, Chien-Yu Lin4, Chung-Yi Hu3, Shiann-Tarng Jou2, Dong-Tsamn Lin1,2, Shu-Rung Lin5,6‡*, Shu-Wha Lin3‡* 1 Departments of Laboratory Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, 2 Departments of Pediatrics, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, 3 Departments of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, 4 Institute of Statistical Science, Academia Sinica, Taipei, Taiwan, 5 Department of Bioscience Technology, College of Science, Chung-Yuan Christian University, Taoyuan, Taiwan, 6 Center for Nanotechnology and Center for Biomedical Technology, College of Science, Chung-Yuan Christian University, Taoyuan, Taiwan OPEN ACCESS Citation: Yang Y-L, Yen C-T, Pai C-H, Chen H-Y, Yu S-L, Lin C-Y, et al. (2015) A Double Negative Loop Comprising ETV6/RUNX1 and MIR181A1 Contributes to Differentiation Block in t(12;21)Positive Acute Lymphoblastic Leukemia. PLoS ONE 10(11): e0142863. doi:10.1371/journal.pone.0142863 Editor: Jörn Lausen, Georg Speyer Haus, GERMANY Received: September 10, 2015 Accepted: October 27, 2015 Published: November 18, 2015 Copyright: © 2015 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the National Science Council of Taiwan, www.most. gov.tw/ (Grant No. NSC 102-2320-B-002-028-MY3, NSC 99-2314-B-002-016-MY3, NSC 100-2632-M033-001-MY3, NSC 101-2632-M-033-001-MY2, NSC 102-2632-M-033-001-MY3, and NSC 102-2320-B033 -003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * (SRL); (SWL) Abstract Childhood acute lymphoblastic leukemia (ALL) with t(12;21), which results in expression of the ETV6/RUNX1 fusion gene, is the most common chromosomal lesion in precursor-B (pre-B) ALL. We identified 17 microRNAs that were downregulated in ETV6/RUNX1+ compared with ETV6/RUNX1− clinical samples. Among these microRNAs, miR-181a-1 was the most significantly reduced (by ~75%; P < 0.001). Using chromatin immunoprecipitation, we demonstrated that ETV6/RUNX1 directly binds the regulatory region of MIR181A1, and knockdown of ETV6/RUNX1 increased miR-181a-1 level. We further showed that miR-181a (functional counterpart of miR-181a-1) could target ETV6/RUNX1 and cause a reduction in the level of the oncoprotein ETV6/RUNX1, cell growth arrest, an increase in apoptosis, and induction of cell differentiation in ETV6/RUNX1+ cell line. Moreover, ectopic expression of miR-181a also resulted in decreased CD10 hyperexpression in ETV6/RUNX1+ primary patient samples. Taken together, our results demonstrate that MIR181A1 and ETV6/RUNX1 regulate each other, and we propose that a double negative loop involving MIR181A1 and ETV6/RUNX1 may contribute to ETV6/RUNX1-driven arrest of differentiation in pre-B ALL. Introduction The t(12;21) translocation, which fuses ETV6 and RUNX1, is the most common chromosomal alteration in childhood precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) [1]. The PLOS ONE | DOI:10.1371/journal.pone.0142863 November 18, 2015 1 / 16 ETV6/RUNX1 and MIR181A1 in ALL Competing Interests: The authors have declared that no competing interests exist. initial fusion of ETV6/RUNX1 is believed to allow quiescent, preleukemic cells to exist in the bone marrow, and the disease-promoting changes in the ETV6/RUNX1-positive preleukemic stage usually take place through second hits that arise in the late pro-B cell stage [2]. The oncogenic property of ETV6/RUNX1 is related to its aberrant function as a rogue transcription factor that can interfere with the normal functions of wild-type ETV6 and RUNX1 through multiple mechanisms. For example, ETV6/RUNX1 can dimerize with wild-type ETV6 via the helix-loop-helix domain of ETV6, thereby disrupting ETV6 function [3, 4]. ETV6/RUNX1 also can bind to RUNX1 target DNA sequences and recruit transcriptional corepressors including mSinA, N-coR, and histone deacetylase-3 (HDAC3) via the ETV6 portion of the fusion protein, resulting in dysregulated RUNX1-dependent transcription [3, 5, 6]. Evidence has revealed that aberrant recruitment of transcriptional repressors correlates with the oncogenic activities of ETV6/RUNX1 so as to constitutively repress a number of genes required for hematopoiesis, including JunD, ACK1, PDGFRB, and TCF4, which are involved in cell cycle regulation [7, 8]. The leukemogenic consequences of ETV6/RUNX1 through the aforementioned mechanisms are induction of survival signals and inhibition of cell differentiation by ETV6/RUNX1’s direct modulation of multiple targets such as EPOR, MDM2, and certain miRNA genes [9–11]. MicroRNAs (miRNAs) execute diverse functions by targeting the mRNAs of multiple genes simultaneously. Recent advances have indicated that miRNAs are important regulators of hematopoiesis; moreover, miRNA-mediated control of gene dosage is critical for lineage fate determination of hematopoietic cells, and disruption of this regulation may lead to malignant transformation [12, 13]. Moreover, dysregulation of miRNA expression is frequently associated with cytogenetic abnormalities, and in turn certain of these abnormalities have a direct impact on aberrant expression of miRNAs [13]. For instance, miR-155 is essential to B-cell development and is aberrantly upregulated in B-cell malignancies including diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL) [14–16]. In patients expressing the aberrant fusion protein AML1/ETO, the most common acute myeloid leukemia–associated fusion resulting from t(8;21), the fusion oncoprotein was the first ever reported to directly repress miR-223 expression by triggering chromatin remodeling and epigenetic silencing, which in turn block myeloid precursor cell differentiation [17]. Emerging evidence from research on miRNAs and hematological malignancy has provided deeper insight into the relation between miRNAs and their target genes. In addition to the simple negative regulation of target mRNAs by miRNAs, miRNA–target relationships may also involve complex feedback and feed-forward loops. These loops help to maintain a desired protein inhibition/ac (...truncated)