Figure 7.

EYA1/SIX1 partially rescues phenotype from abrogated MLL-AF9/BCOR binding and schematic of BCOR/MLL-AF9–dependent gene regulation. A, Survival curves for sublethally irradiated mouse recipients (n = 5–10 per cohort) of BM cells transformed with MLL-AF9 WT (blue), MLL-AF9 E531R (1×; red), MLL-AF9 E531R (NC; red, cross), MLL-AF9 E531R (10×; maroon), MLL-AF9 E531R + Eya1 (green, triangle), or Eya1 alone (green, open diamond). All mice received the same number of transduced cells except those indicated (10×) which received 10-fold more cells. All mice received cells that had been cultured one week in methylcellulose prior to transplant except those indicated as (NC) that had no in vitro culture prior to transplant. Statistical significance compared with MLL-AF9 E531R was determined using a log-rank (Mantel–Cox) test. B, Images of Wright–Giemsa-stained mouse BM cells expressing MLL-AF9, MLL-AF9 E531R, and MLL-AF9 E531R coexpressing Eya1, SIX1, or both Eya1 and SIX1, following the third plating in methylcellulose (scale bar, 10 μm). C, Summary of immunofluorescence staining of MLL-AF9 E531R cells from B for relevant hematopoietic stem and differentiation surface markers. Relative mean fluorescence intensity (MFI) of staining is shown. Data are from three independent experiments; *, P < 0.05. D, Schematic of key effects of loss of BCOR/MLL-AF9 interaction. The levels of expression of Eya1, Six1, Hoxa7, and Cyclins D1–3 are regulated by the interaction of BCOR with WT MLL-AF9. In the absence of the MLL-AF9/BCOR interaction, levels of Eya1 and Six1 are decreased, as is the level of Myc protein, reducing the LIC population and resulting in a lack of in vivo leukemogenesis. The levels of Hoxa7 and Cyclins D1–3 are increased, resulting in increased proliferation as seen in the colony-forming assay.