Identification of Genes Deregulated in Both Human and Murine MLLRearrangement Leukemias.

Abstract

Chromosome translocations are among the most common genetic abnormalities in human leukemia. The mixed lineage leukemia (MLL) gene was identified as a common target of chromosomal translocations associated with human acute leukemias; it is located on human chromosome 11 band q23 and on mouse chromosome 9. More than 50 different loci are rearranged in11q23 leukemias involving MLL, resulting in either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In general, MLL rearrangements are associated with a poor prognosis. MLL-ELL and MLL-ENL resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3) respectively are two common examples of these rearrangements. These two fusions are frequently involved in human AML, while MLL-ENL is also involved in human ALL. There is a common observation that important biological properties are often conserved across species. Cross-species sequence comparison has been widely used to infer gene function, but it is becoming apparent that sequence similarity is not always proportional to functional similarity. To determine the function of a gene precisely, therefore, we need to investigate not only its sequence characteristics but also its expression characteristics. Model organisms have contributed substantially to our understanding of the etiology of human disease and the development of new treatment methodologies. However, although genetically engineered mouse leukemia models have been established for many years, there are few systematic studies to identify and study the genes that exhibit similar abnormal expression patterns in both human leukemia and mouse leukemia model cells. To perform an interspecies gene expression comparative study in leukemia, we used the serial analysis of gene expression (SAGE) technique to examine gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both humans and mice. We obtained 484,303 total SAGE tags for the nine samples and a total of 103,899 unique SAGE tags from five human and 60,993 from four mouse samples. We identified 88 genes that appeared to be significantly deregulated (32 up- and 56 down-regulated) in both human and murine MLL-ELL and/or MLL-ENL leukemia. Fifty-seven genes have not been reported previously. A large-scale quantitative real-time PCR (qPCR) assay was performed to validate the candidate genes, and 84% (36/43) of the tested SAGE candidate genes were confirmed. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9 and HOXA10) and a HOX cofactor MEIS1; their overexpression is a hallmark of MLL-rearrangement leukemia. The top down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI and CYBB. Remarkably, up-regulated genes have a much higher percentage of enrichment in Gene Ontology (GO) terms related to gene expression and transcription, whereas down-regulated genes are more enriched in GO terms related to apoptosis, signal transduction and response. Thus, the up-regulation of genes responsible for gene expression and transcription but down-regulation of genes responsible for apoptosis, signal transduction and response, can promote cell proliferation and inhibit apoptosis, and thereby contribute to the development of leukemia. We showed that the CpG islands of several significantly down-regulated genes including LIF, TGFBI and G0S2 are hypermethylated. We also examined the expression of microRNAs from the mir-17–92 cluster, which are overexpressed in human MLL-rearrangement leukemias, and showed that seven individual microRNAs (i.e., miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92) within this cluster are also overexpressed in mouse MLL-rearrangement leukemia cells. Nineteen putative targets (i.e., APP, RASSF2, SH3BP5, DBN1, ELK3, FLT1, GNAI1, HIF1A, ITGA6, MN1, POU4F1, RB1, RGL1, RNF167, SASH1, SLC24A3, TNFRSF21, WWP1 and YES1) of these microRNAs were reported and/or confirmed by our qPCR to be down-regulated in MLL-rearrangement leukemias. We further confirmed both APP and RASSF2 as direct targets of miR-17 through luciferase reporter and mutagenesis assay. The identification and validation of gene expression changes in MLL-rearrangement human and murine leukemia provides important insights into the genetic pathways that are important for MLL fusion-induced leukemogenesis.