Consistent deregulation of gene expression between human and murine MLL rearrangement leukemias

Abstract

Important biological and pathologic properties are often conserved across species. Although several mouse leukemia models have been well established, the genes deregulated in both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene expression in both human and murine MLL-ELL or MLL-ENL leukemia cells and identified 88 genes that seemed to be significantly deregulated in both types of leukemia cells, including 57 genes not reported previously as being deregulated in MLL-associated leukemias. These changes were validated by quantitative PCR. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) and MEIS1, which are the typical hallmark of MLL rearrangement leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI, and CYBB. Notably, the up-regulated genes are enriched in gene ontology terms, such as gene expression and transcription, whereas the down-regulated genes are enriched in signal transduction and apoptosis. We showed that the CpG islands of the down-regulated genes are hypermethylated. We also showed that seven individual microRNAs (miRNA) from the mir-17-92 cluster, which are overexpressed in human MLL rearrangement leukemias, are also consistently overexpressed in mouse MLL rearrangement leukemia cells. Nineteen possible targets of these miRNAs were identified, and two of them (i.e., APP and RASSF2) were confirmed further by luciferase reporter and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine MLL rearrangement leukemias provide important insights into the genetic base for MLL-associated leukemogenesis.

Figure 1. Expression profiles of candidate genes

(a) Expression profiles of the 88 significant genes from 9 human and mouse samples as detected by SAGE. The genes have at least 3 fold difference in expression value (tag number per 100,000 of total SAGE tag in each library after normalization) between each leukemia sample and the normal control sample (human CD15⁺; mouse: Gr-1⁺), and with a Chi-square test P-value <0.05. See Supplementary Table 1 for details. (b) Expression profies of the 60 significant genes from 20 human and mouse samples as detected by qPCR. Because the number of mouse samples is too small, we combined human and mouse quantitative real-time PCR data together for significance analysis with SAM. Data are presented as ΔC_T. Unsupervised, average linkage hierarchical clustering was performed with Pearson Correlation as distance. All the significant genes have a q value <0.05, with the overall FDR less than 5%. Expression data was mean centered. “_ELL”, MLL-ELL; “_ENL”, MLL-ENL; “_cl”, cell line; “_N”, normal control; “m” or “Mm.”, mouse; “h” or “Hs.”, human; “hm”, human and mouse. In the columns of qRT-PCR, “--”means “no significant change”.

Figure 2. Example of 23 GO terms in which enriched up-regulated genes have an over 2-fold difference of enrichment percentage compared to enriched down-regulated genes

The percentages of the up- and down-regulated genes relative to their total number (i.e., 32 and 55, respectively) in each GO term are shown in the plot for a direct comparison.

Figure 3. DNA methylation analysis of three down-regulated candidate genes (i.e., LTF, TGFBI and G0S2) in MLL-rearrangement leukemias

(a) MSP Assay of CpG islands on the promoter regions of LTF, TGFBI and G0S2. Lanes U, unmethylated products; Lanes M, methylated products. MNC-1 and MNC-2 are two normal controls (bone marrow mononuclear cells). Pos., positive control; Neg., negative control. (b) The CpG islands of LTF, TGFBI and G0S2 were partially demethylated after the treatment with 5-Aza-CdR. Only KOPN1 is the only example shown here (c) Effects of 5-Aza-CdR on the expression of LTF, TGFBI and G0S2 as detected by qPCR. KOPN1, KOCL33 and KOCL51 are ALL cell lines bearing MLL-ENL fusions; KOCL45, KOCL69 and SEM are ALL cell lines bearing MLL-AF4 fusions; KOCL48 and MV4-11 are AML cell lines bearing MLL-AF4 fusions.

Figure 4. Expression profiles and targets of the miRNAs in the mir-17-92 cluster

(a) Expression profiles of the seven miRNAs in 40 human and mouse samples as detected by TaqMan qPCR. All the significant genes have a q value < 0.05, with an overall FDR < 0.05. “_cl” label in the sample ID stands for “cell line”, while “_N” means “normal control” sample; “m”: mouse. (b) Regulation of APP and RASSF2 by miR-17 was confirmed by luciferase reporter and mutagenesis assay. HEK293T cells were co-transfected with an expression construct for miR-17 or an empty vector (control; i.e., MSCVpuro); the luciferase reporter construct containing the wild-type or mutated 3′UTR of APP or RASSF2, along with a β-galactosidase reporter control vector to monitor transfection efficiency. Luciferase was measured 42 hr after transfection. The firefly luciferase activity was normalized to β-galactosidase activity. The normalized luciferase activities represent the firefly: β-galactosidase ratios normalized to the control sample. Error bars present standard deviation obtained from 3 independent experiments. *, p < 0.01 (paired t-test).