A Unique Role of GATA1s in Down Syndrome Acute Megakaryocytic Leukemia Biology and Therapy Ana C. Xavier1., Holly Edwards2,3., Alan A. Dombkowski4,5, Tugce B. Balci6, Jason N. Berman 6 , Graham Dellaire7, Chengzhi Xie2,3, Steven A. Buck1, Larry H. Matherly2,3,8, Yubin Ge2,3, Jeffrey W. Taub1,3,6* 1 Division of Pediatric Hematology/Oncology, Children’s Hospital of Michigan, Detroit, Michigan, United States of America, 2 Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan, United States of America, 3 Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America, 4 Division of Pharmacology and Toxicology, Children’s Hospital of Michigan, Detroit, Michigan, United States of America, 5 Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan, United States of America, 6 IWK Health Centre, Department of Pediatrics, Dalhousie University, Halifax, Canada, 7 Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Canada, 8 Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan, United States of America Abstract Background: Acute megakaryocytic leukemia (AMkL) in Down syndrome (DS) children is uniformly associated with somatic GATA1 mutations, which result in the synthesis of a shorter protein (GATA1s) with altered transactivation activity compared to the wild-type GATA1. It is not fully established whether leukemogenesis and therapeutic responses in DS AMkL patients are due to loss of the wild-type GATA1 or due to a unique function of GATA1s. Methodology: Stable clones of CMK cells with decreased GATA1s or Bcl-2 levels were generated by using GATA1- or BCL-2- specific lentivirus shRNAs. In vitro ara-C, daunorubicin, and VP-16 cytotoxicities of the shRNA stable clones were determined by using the Cell Titer-blue reagent. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Changes in gene transcript levels were determined by gene expression microarray and/or real-time RT-PCR. Changes in protein levels were measured by Western blotting. In vivo binding of GATA1s to IL1A promoter was determined by chromatin immunoprecipitation assays. Results: Lentivirus shRNA knockdown of the GATA1 gene in the DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s), resulting in lower GATA1s protein levels, promoted cell differentiation towards the megakaryocytic lineage and repressed cell proliferation. Increased basal apoptosis and sensitivities to ara-C, daunorubicin, and VP-16 accompanied by down-regulated Bcl-2 were also detected in the CMK GATA1 shRNA knockdown clones. Essentially the same results were obtained when Bcl-2 was knocked down with lentivirus shRNA in CMK cells. Besides Bcl-2, down- regulation of GATA1s also resulted in altered expression of genes (e.g., IL1A, PF4, and TUBB1) related to cell death, proliferation, and differentiation. Conclusion: Our results suggest that GATA1s may facilitate leukemogenesis and potentially impact therapeutic responses in DS AMkL by promoting proliferation and survival, and by repressing megakaryocytic lineage differentiation, potentially by regulating expression of Bcl-2 protein and other relevant genes. Citation: Xavier AC, Edwards H, Dombkowski AA, Balci TB, Berman JN, et al. (2011) A Unique Role of GATA1s in Down Syndrome Acute Megakaryocytic Leukemia Biology and Therapy. PLoS ONE 6(11): e27486. doi:10.1371/journal.pone.0027486 Editor: Eliana Saul Furquim Werneck Abdelhay, Instituto Nacional de Caˆncer, Brazil Received September 28, 2011; Accepted October 18, 2011; Published November 16, 2011 Copyright: 2011 Xavier 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. Funding: This study was supported by grants from the Karmanos Cancer Institute, Children’s Research Center of Michigan, Leukemia Research Life, Children’s Leukemia Foundation of Michigan, National Cancer Institute (CA120772), Elana Fund, the Herrick Foundation, Justin’s Gift Charity, The Buric Family, Leukemia and Lymphoma Society, Sehn Family Foundation, Dale Meyer Memorial Endowment for Leukemia Research, and the Ring Screw Textron Endowed Chair for Pediatric Cancer Research. Microarray data analysis was supported by the Microarray and Bioinformatics Facility Core, Wayne State University, National Institute of Environmental Health Sciences center Grant P30 ES06639. AX is a recipient of a St. Baldrick’s Foundation/Post-Doctoral Fellowship for Childhood Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jtaub@med.wayne.edu . These authors contributed equally to this work. Introduction Down Syndrome (DS) children with leukemia exhibit some of the most unique biological and therapeutic features of leukemia. DS children have an estimated 10-20-fold higher risk for developing acute lymphoblastic leukemia and acute myeloid leukemia (AML) compared to non-DS children.[1] The majority of AML cases in DS children are of the acute megakaryocytic leukemia (AMkL) phenotype.[2–5] It is estimated that DS children have a 500-fold increased risk of developing AMkL compared to non-DS children.[6] Transient myeloproliferative disorder (TMD), a precursor of AMkL, is diagnosed in up to 10% of DS newborns and can resolve spontaneously without chemotherapy in the majority of cases.[7] It is estimated that following clinical resolution, 20–30% of DS TMD patients will subsequently develop AMkL, requiring chemotherapy treatment.[8] Multiple PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27486