Figure 5.
Myb regulates a gene expression program overlapping with MLL-AF9 and associated self-renewal. (A) Heat map of fold change in gene expression for two representative Tet-off-inducible MLL-AF9;Ras leukemia cell lines (on dox relative to off dox) and Tet-on-competent MLL-AF9;Ras leukemia transduced with the indicated Myb shRNAs (shMyb relative to shRen) for all genes with >1.5-fold change in either comparison. (B) Overlap of MLL-AF9 and Myb-regulated gene expression signatures. Using the RRHO approach, genes were rank-ordered according to their differential expression between the indicated subclasses. Spearman rank correlation coefficient = 0.50 (P < 0.001). (C) GSEA plot evaluating LSC-associated genes (Somervaille et al. 2009) after expression of shRNAs targeting Myb (two independent shRNAs, n = 3) or controls (shRen or empty vector, n = 3). (D) GSEA plots evaluating expression of signatures associated with poor prognosis in AML patients in two different studies (Yagi et al. 2003; Metzeler et al. 2008) with or without suppression of Myb. (E) Competitive proliferation assay in MLL-AF9;NrasG12D leukemia. The graph represents the percentage of shRNA-expressing (Venus+dsRed+) cells over time, normalized to initial measurement after 1 d of dox treatment. (F) qRT–PCR analysis for Kit, Myc, and Smyd2 expression in Tet-off-inducible MLL-AF9;Ras leukemia treated with dox for 6 d (relative to off dox) and shRNA-expressing Tet-on-competent MLL-AF9;NrasG12D leukemia transduced with the indicated shRNA, treated with dox for 3 d, and sorted for shRNA-expressing (Venus+dsRed+) cells (relative to shRen). (G) qRT–PCR analysis of MYB, MYC, and SMYD2 in human AML cell lines expressing the indicated shRNAs. Cells were treated with dox for 4 d and sorted for shRNA-expressing (Venus+dsRed+) cells prior to RNA extraction.