Figure 3.

Cross-species transcriptome analysis of iMLL-ENL-derived leukemias revealed common genes that segregate ALL patients with from those without MLL-rearrangements. (A) Volcano plot showing fold change and statistical significance between wt LSK and iMLL-ENL-derived leukemic cells. The 1155 significantly lower expressed genes are plotted in blue and the 780 more highly expressed genes are plotted in red. Cutoffs used for differential expression: FDR < 0.001, absolute log fold change > 2 (same in panels B, C, E, F). (B) Heatmap showing relative expression (log₂ level over mean) of 1935 genes differentially expressed between wt LSK and iMLL-ENL samples. Average expression is shown on the right as logCPM (green). (C) Gene set enrichment analysis (GSEA) results for 2 signatures of the C2 Molecular Signature Database (MsigDB) enriched in the iMLL-ENL/wt LSK signature. Left: “Immortalized by Hoxa9 and Meis1 Up” represents genes upregulated in mouse leukemia by overexpression of Hoxa9 and Meis1 (normalized enrichment score [NES]: 2.0880373; nominal P value: 0.0; FDR q value: 5.326164E−4; family-wise error rate [FWER] P value: 0.006). Right: “Hematopoiesis late progenitor” represents genes upregulated in BM-derived late hematopoietic progenitors (NES: 2.2291334; nominal P value: 0.0; FDR q value: 0.0; FWER P value: 0.0). (D) Hoxa9 and Meis1 mRNA expression measured by qRT-PCR in BM cells isolated from naive wt GMP and wt LSK progenitors and iMLL-ENL or iMLL-AF9 leukemic cell cultured in vitro for 24 hours with DOX or overnight without DOX. Relative expression levels were normalized to Gapdh mRNA levels expression and to expression in naive murine GMP. Results are the mean ± standard deviation of triplicates in the indicated AML groups (n = 3 per group). (E) Pair-wise correlation maps and hierarchical clustering of human patients with the t(11;19) (in purple) and germline MLL (in gray) genotypes. Correlations and clustering of patient samples were computed using expression values of human genes that are orthologous to mouse genes being part of the following 3 gene sets. Left panel: genes significantly differentially expressed between iMLL-ENL cells and wt LSKs. Middle panel: a control set of genes with the same expression distribution as the genes from the iMLL-ENL signature. Right panel: all genes expressed in iMLL-ENL and wt LSK samples. (F) Distribution of normalized expression levels (log₂ CPM) in the 3 gene sets used in (E). ALL = acute lymphoblastic leukemia, AML = acute myeloid leukemia, DOX = doxycycline, GMP = granulocyte-macrophage progenitors, LSK = Lin⁻ Sca-1⁺ c-Kit⁺.