Figure 5

Levels of Lmo2 control proliferation in MLL-ENL transduced mouse leukaemic cells and correlate with overall survival in AML patients. (a) MLL-ENL transduced mouse bone marrow cells show enhanced H3 lysine 9 acetylation at the Lmo2 +1 similar to primary patient samples. Shown is an MVista representation of mouse/human sequence conservation with +1 enhancer highlighted, with a CHIP-on-chip results shown underneath. Annotations are as in Figure 2a. (b) MLL-ENL transduced mouse bone marrow cells show enhanced H3 lysine 9 acetylation across the HoxA cluster similar to primary patient samples. An MVista representation of mouse/human sequence conservation with the ChIP-on-chip results underneath is shown. Annotations are as in Figure 2a. (c) The LMO2 +1 element functions as a transcriptional enhancer in MLL-ENL transduced bone marrow progenitors. Cells were electroporated with luciferase reporter constructs containing the pP, the promoter together with the wild-type +1 enhancer, or the promoter with two different mutant versions of +1 enhancer. Mean and s.e.m. for at least two independent transfections (each one performed in triplicate) are shown. Values are expressed relative to the vector containing the luciferase gene under the control of the minimal pP alone (pP-Luc). (d) Knock-down of Lmo2 in MLL-ENL transduced cells results in a competitive growth disadvantage. MLL-ENL immortalized bone marrow progenitors were transduced with constructs containing shRNA against Lmo2 (triangles) or luciferase (squares) as a control. GFP presence was monitored over 25 days after infection and percentages of GFP-positive cells are indicated. The results from a representative experiment performed in duplicate are shown. (e) Overall survival over 3 years in a cohort of 79 AML patients shows statistically significant association with levels of LMO2 expression at diagnosis. Shown is a boxplot of expression levels for patients with overall survival of less (left) and more (right) than 3 years, with the corresponding P-value shown at the top right. (f) Lower LMO2 expression at diagnosis shows a strong association with improved survival. Kaplan–Meier survival curves for AML patients with log 2 expression scores for LMO2 higher than 11.5 (blue curve) or lower than 11.5 (red curve) at diagnosis are shown. Corresponding P-value is also shown.