Figure 5.

Selective requirement for Pip4k2a in murine MLL-AF9 AML cells. Murine normal KIT⁺ or MLL-AF9 AML cells were infected with lentiviral vectors targeting Pip4k2a for KD, or a non-targeting control (NTC), with eGFP as the selectable marker. GFP⁺ cells were FACS purified 48 hours following lentiviral infection and initiation of KD. (a) Bar chart shows mean±SEM (of triplicate analyses) expression of Pip4k2a relative to control MLL-AF9 AML cells for the indicated normal and leukemic populations, as determined by quantitative PCR, 48 hours following initiation of KD. (b) Bar chart shows mean±SEM colony forming cell (CFC) frequencies (n=3) of control and Pip4k2a KD MLL-AF9 AML cells. * indicates p<0.05 by comparison with control cells, as determined by one way ANOVA followed by Fisher’s least significant difference post hoc test. (c) Representative cell cycle profiles show the proportion of MLL-AF9 AML cells in each stage of the cell cycle, or sub-G₁ apoptotic cells, in control or Pip4k2a KD cells 96 hours following initiation of KD. (d) Excluding sub-G₁ cells and related to (c), table indicates mean±SEM percentage of murine MLL-AF9 AML cells in the indicated phase of the cell cycle (n=3 triplicate analyses). * indicates p<0.05 by comparison with control cells, as determined by one way ANOVA followed by Fisher’s least significant difference post hoc test. (e) Bar chart shows mean±SEM colony forming cell (CFC) frequencies (n=3) of control and Pip4k2a KD KIT⁺ normal HSPC. (f) Heat map represents 421 protein coding genes significantly up or down regulated (p<0.001 (unpaired t-test) and more than two-fold change in expression) in MLL-AF9 AML cells by comparison with KIT⁺ HSPC, with key genes highlighted. Color scale indicates normalized expression values. Venn diagrams indicate numbers of protein coding genes up or down regulated (defined as those whose expression increased or decreased by 25% compared with control samples) following Pip4k2a KD for each of the tested KD constructs (#1 and #2) in (g) MLL-AF9 AML cells and (h) KIT⁺ BM HSPC. The number of co-regulated genes is shown in the intersect, together with the significance of the overlap (Chi-square test). (i) Venn diagrams indicate extent of overlap of genes up or down regulated by Pip4k2a KD in MLL-AF9 AML cells (M9) or KIT+ BM HSPC (KIT).