Figure 7.

Nras^G12D cooperates with MOL4070LTR to induce AML. (A) Kaplan-Meier survival curves of WT and Mx1-Cre, Nras^G12D pups that were injected with MOL4070LTR (+MOL) and of littermate controls that did not receive this virus (−MOL). Mx1-Cre, Nras^G12D mice in the +MOL cohort show significantly shorter survival than WT littermates +MOL (P < .0001) or the Mx1-Cre, Nras^G12D −MOL group ( < .0001). (B) Blood smear from a Mx1-Cre, Nras^G12D mouse with AML. Slides were examined by Nikon Eclipse E400 microscope with 40×/0.75 NA oil objective. Picture was taken with Nikon Coolpix 5000 camera and analyzed with Adobe Photoshop CS3. (C) Flow cytometry demonstrating Mac1 and Gr1 expression on myeloblasts. (D) Southern blot analysis with a probe to the MOL4070 LTR sequences demonstrates that Mx1-Cre, Nras^G12D leukemias are clonal. Primary AMLs (1, 2, and 3) were transplanted into sublethally irradiated recipients (labeled as 1T1, etc). (E) Semiquantitative PCR with primers that amplify both WT and mutant Nras allele demonstrates the loss of the WT allele in most Nras mutant AMLs (the amplification product corresponding to the WT Nras allele is not visible in lanes 1 and 2, and greatly reduced in intensity in lanes 3, 5, and 6). (F) Evi1 expression measured by quantitative reverse-transcribed PCR in WT whole bone marrow (WT), AML that does not have Evi1 integration (Neg), and 3 AMLs that harbor Evi1 integrations (Pos1, Pos2, and Pos3). The level of Evi1 was normalized to expression of GAPDH and presented as fold increase compared with the level in the WT whole bone marrow. Error bars represent SD from triplicates. (G) Blast colony growth from Nras mutant AMLs (red line) is hypersensitive to the MEK inhibitor PD0325901 compared with CFU-GM colony growth from WT marrow (blue line). Colony growth was assayed in methylcellulose cultures containing a saturating dose of GM-CSF (10 ng/mL) over a range of PD0325901 concentrations.