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Consistent Deregulation of Gene Expression Between Human and Murine MLL Rearrangement Leukemias

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Consistent Deregulation of Gene Expression Between Human and Murine MLL Rearrangement Leukemias Published OnlineFirst January 20, 2009; DOI: 10.1158/0008-5472.CAN-08-3381 Research Article Consistent Deregulation of Gene Expression between Human and Murine MLL Rearrangement Leukemias Zejuan Li,1 Roger T. Luo,1 Shuangli Mi,1 Miao Sun,1 Ping Chen,1 Jingyue Bao,2 Mary Beth Neilly,1 Nimanthi Jayathilaka,1 Deborah S. Johnson,3 Lili Wang,2 Catherine Lavau,4 Yanming Zhang,1 Charles Tseng,3 Xiuqing Zhang,2 Jian Wang,2 Jun Yu,2 Huanming Yang,2 San Ming Wang,5 Janet D. Rowley,1 Jianjun Chen,1 and Michael J. Thirman1 1Department of Medicine, University of Chicago, Chicago, Illinois; 2Beijing Genomics Institute, Chinese Academy of Sciences, Beijing, China; 3Department of Biological Sciences, Purdue University Calumet, Hammond, Indiana; 4CNRS UPR9051, IUH, Paris, France; and 5Center for Functional Genomics, Division of Medical Genetics, Department of Medicine, ENH Research Institute, Northwestern University, Evanston, Illinois Abstract translocations associated with human acute leukemias (1, 2). MLL Important biological and pathologic properties are often is located on human chromosome 11 band q23 and on mouse conserved across species. Although several mouse leukemia chromosome 9. More than 50 different loci are rearranged models have been well established, the genes deregulated in in11q23 leukemias involving MLL in either acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL; ref. 3). both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene MLL rearrangements are associated with a poor prognosis (4). expression in both human and murine MLL-ELL or MLL-ENL MLL-ELL and MLL-ENL that result from t(11;19)(q23;p13.1) and leukemia cells and identified 88 genes that seemed to be t(11;19)(q23;p13.3), respectively (1, 5), are two common examples of significantly deregulated in both types of leukemia cells, these rearrangements. These two fusions are frequently involved in including 57 genes not reported previously as being deregu- human AML, whereas MLL-ENL is also involved in human ALL. lated in MLL-associated leukemias. These changes were The translocations result in an in-frame fusion of the NH2 terminus validated by quantitative PCR. The most up-regulated genes of the MLL gene and the COOH terminus of each partner gene. include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) Retroviral-mediated gene transfer of MLL-ENL and MLL-ELL and MEIS1, which are the typical hallmark of MLL rearrange- transforms primary myeloid progenitor cells and cause acute myeloid leukemia in mice (6, 7). ment leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI,andCYBB. Gene expression profiles differ between distinct subtypes of Notably, the up-regulated genes are enriched in gene ontology leukemia and provide specific markers for clinical diagnosis. It is terms, such as gene expression and transcription, whereas the commonly observed that important biological/pathologic proper- down-regulated genes are enriched in signal transduction and ties are often conserved across species (8, 9). Model organisms have apoptosis. We showed that the CpG islands of the down- contributed substantially to our understanding of the etiology of regulated genes are hypermethylated. We also showed that human disease and the development of new treatment method- seven individual microRNAs (miRNA) from the mir-17-92 ologies (10). However, although genetically engineered mouse cluster, which are overexpressed in human MLL rearrange- leukemia models have been well established (6, 7, 11, 12), there are ment leukemias, are also consistently overexpressed in mouse few systematic studies to identify and study the genes that exhibit MLL rearrangement leukemia cells. Nineteen possible targets similar abnormal expression patterns in both human and murine of these miRNAs were identified, and two of them (i.e., APP leukemia cells. To perform an interspecies gene expression and RASSF2) were confirmed further by luciferase reporter comparative study in leukemia, we used the serial analysis of gene expression (SAGE) technique (13) to compare gene expression and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells MLL rearrangement leukemias provide important insights and normal myeloid progenitor cells in both humans and mice. into the genetic base for MLL-associated leukemogenesis. Herein, we report the identification and validation of differentially [Cancer Res 2009;69(3):1109–16] expressed genes commonly present in both human and murine MLL-ELL or MLL-ENL leukemias. Introduction Chromosome translocations are among the most common Materials and Methods genetic abnormalities in human leukemia. The MLL (mixed lineage Patient samples. The patient samples were obtained at the time of leukemia) gene was identified as a common target of chromosomal diagnosis and with informed consent at University of Chicago and were stored in liquid nitrogen until use. SAGE assay and data analysis. Cell purification, total RNAisolation, cDNAsynthesis, and SAGEwere carried out according to our established Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). procedures (13–15). SAGE tag sequences were extracted with SAGE 2000 S.M. Wang, J.D. Rowley, J. Chen, and M.J. Thirman contributed equally to this work. software. Tag counts were converted to counts per 100,000, and the Requests for reprints: J. Chen, J.D. Rowley, or M.J. Thirman, 5841 South Maryland expression data were cross-linked to UniGene clusters by extracting the Avenue, MC 2115, Chicago, IL 60637. E-mail: [email protected], 3¶-most NlaIII SAGE tag for each transcript in each UniGene clusters. Only [email protected], or [email protected]. I2009 American Association for Cancer Research. tags that matched to a single gene cluster were taken into account. All doi:10.1158/0008-5472.CAN-08-3381 SAGE tags mapped to the same gene were then combined, and the sum of www.aacrjournals.org 1109 Cancer Res 2009; 69: (3).February 1, 2009 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst January 20, 2009; DOI: 10.1158/0008-5472.CAN-08-3381 Cancer Research their counts in a given sample represented the expression level/signal of ELL or MLL-ENL fusions and a leukemic mouse cell line with an 2 that gene in that sample (16, 17). m test was used to identify differentially MLL-ELL fusion (Table 1). SAGE tags (484,303) were identified m2 expressed genes, as Man and colleagues (18) showed that test has the from the nine samples (total SAGE tag counts of each sample best power and robustness in SAGE data analysis. varies from 40,000 to 100,000, with 53,811 tags per sample on Functional classification and annotation of the candidate genes. We average), yielding 103,899 unique tags in human and 60,993 in used the Database for Annotation, Visualization, and Integrated Discovery (DAVID; version 2008)6 for functional classification and annotation of mouse samples (see Table 1). candidate genes. DAVID provides a comprehensive set of functional We identified 88 candidate genes that seemed to be deregulated annotation tools for investigators to understand the biological meaning in both human and murine MLL-ELL and/or MLL-ENL leukemias. behind large list of genes (19). Agene cluster means a set of genes belonging Of them, 6 and 26 genes are up-regulated and down-regulated, to the same functional category, as defined by DAVID. respectively, in both types of leukemia; 3 and 13 genes are up- Quantitative real-time PCR assays of protein coding or microRNA regulated and down-regulated, respectively, in MLL-ELL leukemias genes and data analysis. We performed quantitative real-time PCR (qPCR) alone; 23 and 17 genes are up-regulated and down-regulated, to determine expression of protein coding genes with SYBR green dye using respectively, in MLL-ENL leukemias alone (see Fig. 1A and kits from Qiagene and microRNAs (miRNA) using TaqMan qPCR kits from Supplementary Table S1). All the significant genes have at least Applied Biosystems. PGK1 or glyceraldehyde-3-phosphate dehydrogenase 3-fold difference in expression (tag counts per 100,000) between was used as endogenous controls for protein coding genes, whereas U6 RNA m2 was used as an endogenous control for miRNAs. PCR reactions and data each leukemia sample and the normal control and with a test analyses were performed, as described previously (20, 21). P value of <0.05. As this criterion is stringent, it therefore limits the Methylation-specific PCR. The methylation status of the promoter total number of the candidate genes identified. region was determined by the methylation-specific PCR (MSP) method (22). Notably, the four most up-regulated genes (each has at least a The primers were designed to anneal specifically to methylated and 30-fold change in expression) in both types of human and mouse unmethylated CpG dinucleotides in promoter regions of genes using the leukemia samples are three homeobox (HOX) genes (i.e., HOXA5, Primer3 program. Genomic DNAwas isolated using QIAampDNAmini kit HOXA9, and HOXA10) and one HOX cofactor, MEIS1 (see Fig. 1A) (Qiagen). One microgram of DNAwas used for bisulfite modification using The eight most down-regulated genes (each has at least a 30-fold the CpGenome DNAmodification kit (Chemicon) according to the change in expression on average) in both types of human and manufacturer’s instructions. The bisulfite-converted DNAwas amplified in mouse leukemia samples include LTF, LCN2, MMP9, S100A8, a total volume of 20 AL using GeneAmp Gold buffer containing 4 mmol/L A S100A9, PADI4, TGFBI, and CYBB (see Supplementary Table S1). MgCl2,0.5 mol/L of each primer, 0.2 mmol/L deoxynucleotide triphos- phate, 5 Ag bovine serum albumin, and 1.25 units of AmpliTaq Gold DNA Note that the fold change of some up-regulated or down-regulated polymerase (Roche).
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