Figure 1.

Mutant Npm1 cooperates with Nras-G12D and Flt3-ITD to enhance myeloid differentiation and enhance progenitor self-renewal. (A) Schema for Mx-1 Cre, Npm1^flox-cA, Nras^LSL-G12D, and Flt3^ITD intercrosses. (B) Nras^G12D/+ mice show a subtle and Npm1^cA/+;Flt3^ITD/+ mice a marked increase in WCC, in comparison with wild-type. Splenic sizes were significantly increased in all mutant genotypes except Npm1^cA/+, with Npm1^cA/+;Flt3^ITD/+ showing the most striking phenotype. Bone marrow cellularity was increased only in the presence of the Nras^G12D/+ allele. (C) FACS analysis at 4 to 5 weeks after mutation induction. Gating strategies depicted are from wild-type mice. There were significant differences in the stem and progenitor cell compartments of Nras^G12D/+ and Flt3^ITD/+, but not of Npm1^cA/+ single-mutant, mice, as has been previously reported. In double-mutant mice, the Npm1^cA/+;Nras^G12D/+ combination was not significantly different to Nras^G12D/+, in contrast to Npm1^cA/+;Flt3^ITD/+, which was markedly different from both Flt3^ITD/+ and Npm1^cA/+ single mutants. (D) Using a cell surface phenotype independent of FLT3 staining, we found that CD45⁺/EPCR⁺/CD150⁺/CD48⁻ HSCs were reduced slightly in Npm1^cA/+;Nras^G12D/+ and markedly in Npm1^cA/+;Flt3^ITD/+ mice. (E) Summary of hematopoietic effects of Npm1^cA/+;Nras^G12D/+ and Npm1^cA/+;Flt3^ITD/+ double mutations in mice. (F) Single Npm1^cA/+- and double Npm1^cA/+;Nras^G12D/+- or Npm1^cA/+;Flt3^ITD/+-mutant hematopoietic progenitors show increased self-renewal potential in whole bone marrow serial replating assays (n = 4-8). Mean ± SEM are plotted. Significant values are reported for 1-way analysis of variance (Bonferroni adjusted). LT, long-term; Prog., progenitors; ST, short-term. *P < .05, **P < .01, ***P < .001, all vs wild-type; ^ΔP < .05, ^ΔΔΔP < .001, all vs Flt3^ITD/+; ^♣♣♣P < .001, all vs Nras^G12D/+; ^†P < .05, ^††P < .01, ^†††P < .001, all Npm1^cA/+;Nras^G12D/+ vs Npm1^cA/+;Flt3^ITD/+. WCC, white cell count.