Coexpression of NPM1c and FLT3-D835Y induces a STAT5-mediated rapid onset MPN in mice. (A) Kaplan-Meier plots for survival of recipients of 25 000 Npm^{flox cA/+} or Npm1^+/+ BM cells transduced with FLT3-D835Y, FLT3-ITD, or empty vector (MiG) showing the rapid onset of leukemic disease in Flt3-D835Y⁺Npm1c⁺ mice. A total of 26 mice (from 4 independent transplantations) are presented that received Flt3-D835Y⁺Npm1^{flox cA/+}Mx1-Cre BM. Seven mice received Flt3-ITD⁺Npm1^{flox cA/+}Mx1-Cre BM, 5 mice Flt3-ITD⁺Npm1^+/+Mx1-Cre BM, 13 mice received Flt3-D835Y⁺Npm1^{flox cA/+} BM in the absence of Mx1-Cre, 5 mice received Flt3-D835Y⁺Npm1^+/+Mx1Cre BM, and 5 mice received MiG Npm1^{flox cA/+}Mx1-Cre BM. (B) Percentage of PB EGFP⁺ cells at indicated times demonstrating increased leukemic burden in Flt3-D835Y⁺Npm1c⁺ mice compared with Flt3-D835Y⁺Npm1 wt mice (21 days posttransplant: n = 28 [NPM1c], n = 17 [NPM1 wt]; 33 days posttransplant: n = 12 [NPM1c], n = 7 [NPM1 wt]; 48 days posttransplant: n = 7 [NPM1c], n = 15 [NPM1 wt]). (C) Fold induction of BM EGFP⁺ cells shows a significantly enhanced leukemic burden in Flt3-D835Y⁺Npm1c⁺ moribund mice (n = 22) compared with Flt3-D835Y⁺Npm1 wt mice (n = 7; sacrificed after more than 130 days). (D) Increased spleen weights of Flt3-D835Y⁺Npm1c⁺ moribund mice (n = 22) compared with Flt3-D835Y⁺Npm1 wt group; (n = 6; sacrificed after more than 130 days). (E) CD45⁺ BM cells from a representative mouse were stained for Gr-1, B220, or Thy1.2 and analyzed by flow cytometry. Numbers indicate percentage of cells in the respective quadrant gate. Flt3-D835Y⁺Npm1c⁺Mx1-Cre mice display a phenotypic shift to a myeloproliferative disease with elevated numbers of EGFP⁺/Gr-1⁺ cells. Phenotype distributions are shown in supplemental Table 1 and supplemental Figure 1. (F) Immunofluorescence staining of pSTAT5 in EGFP⁺ BM cells from moribund mice showing STAT5 activation only in Flt3-D835Y⁺Npm1c⁺ cells. (G) Quantification of the percentage of positive cells in n = 9 (NPM1c) and n = 4 (NPM1 wt) pictures. (H) Immunofluorescence staining of pSTAT3 in EGFP⁺ BM cells. pSTAT3 is exclusively present in Flt3-D835Y⁺Npm1 wt cells. (I) Quantification of the percentage of positive cells in n = 7 (NPM1c) and n = 4 (NPM1 wt) pictures. (J) Splenocytes from moribund mice were analyzed for STAT5, STAT3, and ERK1/2 signaling by immunoblotting. Flt3-D835Y⁺Npm1c⁺ cells display a strong induction of STAT5 signaling and little STAT3 activation. Flt3-D835Y⁺Npm1 wt cells show no STAT5 activation, but activation of STAT3. (K) Flow cytometric analysis of pSTAT5 and pSTAT3 in EGFP⁺ BM cells from moribund mice confirming the activation of STAT5 in Flt3-D835Y⁺Npm1c⁺ cells, but not in Flt3-D835Y⁺Npm1 wt cells. *P < .05, **P < .01, and ***P < .001, by unpaired, 2-tailed Student t test (panels B-D, G, and I) or logrank test (panel A).