Figure 5.
Heterozygous Hippo kinase inactivation cooperates with JAK2-V617F to promote lethal MF in mice. Genotypes used in this model are Stk4f/+Stk3f/+;Vav1-Cre+ (Stk4+/−Stk3+/−) and Stk4f/+Stk3f/+; Vav1-Cre− (Stk4+/+Stk3+/+). (A) Experimental schematic demonstrating the strategy for JAK2-V617F retroviral transduction/transplantation murine model of MPN. Mice were monitored up 36 weeks after transplant. Fluorouracil (5-FU)-treated bone marrow was pooled from 9 mice per genotype per transduction/transplantation experiment. Total mice analyzed in this model were as follows: n = 11 (Vector-Stk4+/+Stk3+/+), n = 10 (Vector-Stk4+/−Stk3+/−), n = 14 (V617F-Stk4+/+Stk3+/+), and n = 15 (V617F-Stk4+/−Stk3+/−). (B) Peripheral blood GFP+ (JAK2-V617F expressing) cell frequencies at the indicated time points measured via flow cytometry: n = 14 (V617F-Stk4+/+Stk3+/+) and n = 15 (V617F-Stk4+/−Stk3+/−). Data are means ± SEM. (C) Peripheral blood hematocrit (%) measurements at the indicated time points: n = 11 (Vector-Stk4+/+Stk3+/+), n = 10 (Vector-Stk4+/−Stk3+/−), n = 7 (V617F-Stk4+/+Stk3+/+), and n = 8 (V617F-Stk4+/−Stk3+/−). (D) As in panel C, but for platelet counts. (E) As in panel C, but for granulocyte counts. (F) Kaplan-Meier survival plot indicating overall survival for the indicated groups over the course of 36 weeks. Statistical significance is determined by log rank (Mantel-Cox) test: n = 11 (vector-Stk4+/+Stk3+/+), n = 10 (vector-Stk4+/−Stk3+/−), n = 14 (V617F-Stk4+/+Stk3+/+), and n = 15 (V617F-Stk4+/−Stk3+/−). (G) Spleen weights for mice of the indicated groups analyzed at the 36-week end point or upon euthanasia for disease progression: n = 11 (vector-Stk4+/+Stk3+/+), n = 10 (vector-Stk4+/−Stk3+/−), n = 13 (V617F-Stk4+/+Stk3+/+), and n = 14 (V617F-Stk4+/−Stk3+/−). Statistical significance was determined by one-way ANOVA followed by the post hoc Tukey test for multiple comparisons. (H) Quantification of MF grades 0-3 in murine bone marrow sections 36 weeks after transplant, based on an established fibrosis grading scale (see methods). Statistical significance between V617F groups was determined by 2-tailed Student t test. (I) Images of representative H&E-stained (top) and reticulin-stained (bottom) sections from the 3 groups indicated. Vector are representative of grade 0 MF, V617F-Stk4+/+Stk3+/+ is representative of grades 0 to 1 MF, and V617F-Stk4+/−Stk3+/− is representative of grades 1 to 2 MF. Scale bar, 100 µm. Data from 2 independent transduction/transplantation experiments with similar group size that demonstrated comparable results are combined. Data are means ± SEM. *P < .05, **P < .01.

Heterozygous Hippo kinase inactivation cooperates with JAK2-V617F to promote lethal MF in mice. Genotypes used in this model are Stk4f/+Stk3f/+;Vav1-Cre+ (Stk4+/−Stk3+/−) and Stk4f/+Stk3f/+; Vav1-Cre− (Stk4+/+Stk3+/+). (A) Experimental schematic demonstrating the strategy for JAK2-V617F retroviral transduction/transplantation murine model of MPN. Mice were monitored up 36 weeks after transplant. Fluorouracil (5-FU)-treated bone marrow was pooled from 9 mice per genotype per transduction/transplantation experiment. Total mice analyzed in this model were as follows: n = 11 (Vector-Stk4+/+Stk3+/+), n = 10 (Vector-Stk4+/−Stk3+/−), n = 14 (V617F-Stk4+/+Stk3+/+), and n = 15 (V617F-Stk4+/−Stk3+/−). (B) Peripheral blood GFP+ (JAK2-V617F expressing) cell frequencies at the indicated time points measured via flow cytometry: n = 14 (V617F-Stk4+/+Stk3+/+) and n = 15 (V617F-Stk4+/−Stk3+/−). Data are means ± SEM. (C) Peripheral blood hematocrit (%) measurements at the indicated time points: n = 11 (Vector-Stk4+/+Stk3+/+), n = 10 (Vector-Stk4+/−Stk3+/−), n = 7 (V617F-Stk4+/+Stk3+/+), and n = 8 (V617F-Stk4+/−Stk3+/−). (D) As in panel C, but for platelet counts. (E) As in panel C, but for granulocyte counts. (F) Kaplan-Meier survival plot indicating overall survival for the indicated groups over the course of 36 weeks. Statistical significance is determined by log rank (Mantel-Cox) test: n = 11 (vector-Stk4+/+Stk3+/+), n = 10 (vector-Stk4+/−Stk3+/−), n = 14 (V617F-Stk4+/+Stk3+/+), and n = 15 (V617F-Stk4+/−Stk3+/−). (G) Spleen weights for mice of the indicated groups analyzed at the 36-week end point or upon euthanasia for disease progression: n = 11 (vector-Stk4+/+Stk3+/+), n = 10 (vector-Stk4+/−Stk3+/−), n = 13 (V617F-Stk4+/+Stk3+/+), and n = 14 (V617F-Stk4+/−Stk3+/−). Statistical significance was determined by one-way ANOVA followed by the post hoc Tukey test for multiple comparisons. (H) Quantification of MF grades 0-3 in murine bone marrow sections 36 weeks after transplant, based on an established fibrosis grading scale (see methods). Statistical significance between V617F groups was determined by 2-tailed Student t test. (I) Images of representative H&E-stained (top) and reticulin-stained (bottom) sections from the 3 groups indicated. Vector are representative of grade 0 MF, V617F-Stk4+/+Stk3+/+ is representative of grades 0 to 1 MF, and V617F-Stk4+/−Stk3+/− is representative of grades 1 to 2 MF. Scale bar, 100 µm. Data from 2 independent transduction/transplantation experiments with similar group size that demonstrated comparable results are combined. Data are means ± SEM. *P < .05, **P < .01.

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