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Loss of wild-type Jak2 allele enhances myeloid cell expansion and accelerates myelofibrosis in Jak2V617F knock-in mice.

Akada H, Akada S, Hutchison RE, Mohi G - Leukemia (2014)

Bottom Line: JAK2V617F homozygosity via 9pLOH has been associated with more severe MPN phenotype.Together, these results suggest that loss of Jak2 WT allele increases the severity of the MPN.Thus, the Jak2 WT allele functions as a negative regulator of MPN induced by Jak2V617F.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, USA.

ABSTRACT
JAK2V617F is the most common mutation found in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p (9pLOH) involving the JAK2 locus has been observed in ∼30% of MPN patients. JAK2V617F homozygosity via 9pLOH has been associated with more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the roles of wild-type JAK2 (JAK2 WT) and JAK2V617F alleles in the development of MPNs, we have used conditional Jak2 knock-out and Jak2V617F knock-in mice and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like MPN in mice, loss of Jak2 WT allele in hemizygous or homozygous Jak2V617F mice results in markedly increased white blood cells, neutrophils, reticulocytes and platelets in the peripheral blood, and significantly larger spleen size compared with heterozygous Jak2V617F mice. Hemizygous or homozygous Jak2V617F mice also exhibit accelerated myelofibrosis compared with mice expressing heterozygous Jak2V617F. Together, these results suggest that loss of Jak2 WT allele increases the severity of the MPN. Thus, the Jak2 WT allele functions as a negative regulator of MPN induced by Jak2V617F.

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Deletion of Jak2 WT enhances constitutive signaling in primary hematopoietic cells expressing Jak2V617FPrimary megakaryoblasts (a) and erythroblasts (b) were derived from the BM of control, heterozygous (Jak2VF/+), hemizygous (Jak2VF/−) and homozygous (Jak2VF/VF) Jak2V617F mice. Cells were serum and factor depleted for 6 hours and lysed in RIPA buffer. Immunoblotting was performed using phospho-specific antibodies against Stat5, Akt and Erk1/2. Membranes were re-probed with respective total antibodies. Total Jak2 protein levels were also determined by immunoblotting using anti-Jak2 antibody. β-actin was used as a loading control. Histograms (in the right panels) demonstrate the fold changes in phosphorylation of Stat5, Akt and Erk1/2 when compared to the phosphorylation levels of those proteins in heterozygous Jak2V617F-expressing megakaryoblasts or erythroblasts in the absence of cytokines. Data from three to four independent experiments are shown in histograms as mean ± SEM. Asterisks indicate significant differences (p < 0.05). Notably, deletion of Jak2 WT allele enhanced constitutive phosphorylation/activation of Stat5, Erk1/2 and/or Akt in primary hematopoietic cells from hemizygous and homozygous Jak2V617F mice.
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Figure 6: Deletion of Jak2 WT enhances constitutive signaling in primary hematopoietic cells expressing Jak2V617FPrimary megakaryoblasts (a) and erythroblasts (b) were derived from the BM of control, heterozygous (Jak2VF/+), hemizygous (Jak2VF/−) and homozygous (Jak2VF/VF) Jak2V617F mice. Cells were serum and factor depleted for 6 hours and lysed in RIPA buffer. Immunoblotting was performed using phospho-specific antibodies against Stat5, Akt and Erk1/2. Membranes were re-probed with respective total antibodies. Total Jak2 protein levels were also determined by immunoblotting using anti-Jak2 antibody. β-actin was used as a loading control. Histograms (in the right panels) demonstrate the fold changes in phosphorylation of Stat5, Akt and Erk1/2 when compared to the phosphorylation levels of those proteins in heterozygous Jak2V617F-expressing megakaryoblasts or erythroblasts in the absence of cytokines. Data from three to four independent experiments are shown in histograms as mean ± SEM. Asterisks indicate significant differences (p < 0.05). Notably, deletion of Jak2 WT allele enhanced constitutive phosphorylation/activation of Stat5, Erk1/2 and/or Akt in primary hematopoietic cells from hemizygous and homozygous Jak2V617F mice.

Mentions: To further confirm that wild-type Jak2 acts as a negative regulator of hematopoietic signaling mediated by Jak2V617F, we compared the effects of heterozygous, hemizygous and homozygous Jak2V617F expression on hematopoietic signaling. Primary megakaryoblasts and erythroblasts derived from the BM of control (Jak2+/+), heterozygous (Jak2VF/+), hemizygous (Jak2VF/−) and homozygous Jak2V617F (Jak2VF/VF) mice were utilized for signaling studies. Whereas heterozygous Jak2V617F expression resulted in constitutive activation of Stat5, Erk1/2 and/or Akt, hemizygous or homozygous Jak2V617F expression further enhanced the constitutive activation of these signaling pathways (Figures 6a and b). Homozygous Jak2V617F-expressing megakaryoblasts/erythroblasts exhibited the highest level of activation of Stat5, Erk1/2 and Akt signaling (Figures 6a and b). Hemizygous Jak2V617F megakaryoblasts and erythroblasts, which harbored only one allele of Jak2 (Jak2V617F) and thus expressed lower levels of total Jak2 protein than in control, heterozygous or homozygous Jak2V617F megakaryoblasts/erythroblasts, showed greater activation of constitutive signaling compared with control or heterozygous Jak2V617F megakaryoblasts/erythroblasts (Figures 6a and b). Notably, activation of Stat5 and Erk1/2 was much greater in megakaryoblasts than in erythroblasts of hemizygous Jak2V617F mice compared with those in heterozygous Jak2V617F mice (Figures 6a and b). Overall, these results suggest that wild-type Jak2 negatively regulates signaling mediated by Jak2V617F.


Loss of wild-type Jak2 allele enhances myeloid cell expansion and accelerates myelofibrosis in Jak2V617F knock-in mice.

Akada H, Akada S, Hutchison RE, Mohi G - Leukemia (2014)

Deletion of Jak2 WT enhances constitutive signaling in primary hematopoietic cells expressing Jak2V617FPrimary megakaryoblasts (a) and erythroblasts (b) were derived from the BM of control, heterozygous (Jak2VF/+), hemizygous (Jak2VF/−) and homozygous (Jak2VF/VF) Jak2V617F mice. Cells were serum and factor depleted for 6 hours and lysed in RIPA buffer. Immunoblotting was performed using phospho-specific antibodies against Stat5, Akt and Erk1/2. Membranes were re-probed with respective total antibodies. Total Jak2 protein levels were also determined by immunoblotting using anti-Jak2 antibody. β-actin was used as a loading control. Histograms (in the right panels) demonstrate the fold changes in phosphorylation of Stat5, Akt and Erk1/2 when compared to the phosphorylation levels of those proteins in heterozygous Jak2V617F-expressing megakaryoblasts or erythroblasts in the absence of cytokines. Data from three to four independent experiments are shown in histograms as mean ± SEM. Asterisks indicate significant differences (p < 0.05). Notably, deletion of Jak2 WT allele enhanced constitutive phosphorylation/activation of Stat5, Erk1/2 and/or Akt in primary hematopoietic cells from hemizygous and homozygous Jak2V617F mice.
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Related In: Results  -  Collection

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Figure 6: Deletion of Jak2 WT enhances constitutive signaling in primary hematopoietic cells expressing Jak2V617FPrimary megakaryoblasts (a) and erythroblasts (b) were derived from the BM of control, heterozygous (Jak2VF/+), hemizygous (Jak2VF/−) and homozygous (Jak2VF/VF) Jak2V617F mice. Cells were serum and factor depleted for 6 hours and lysed in RIPA buffer. Immunoblotting was performed using phospho-specific antibodies against Stat5, Akt and Erk1/2. Membranes were re-probed with respective total antibodies. Total Jak2 protein levels were also determined by immunoblotting using anti-Jak2 antibody. β-actin was used as a loading control. Histograms (in the right panels) demonstrate the fold changes in phosphorylation of Stat5, Akt and Erk1/2 when compared to the phosphorylation levels of those proteins in heterozygous Jak2V617F-expressing megakaryoblasts or erythroblasts in the absence of cytokines. Data from three to four independent experiments are shown in histograms as mean ± SEM. Asterisks indicate significant differences (p < 0.05). Notably, deletion of Jak2 WT allele enhanced constitutive phosphorylation/activation of Stat5, Erk1/2 and/or Akt in primary hematopoietic cells from hemizygous and homozygous Jak2V617F mice.
Mentions: To further confirm that wild-type Jak2 acts as a negative regulator of hematopoietic signaling mediated by Jak2V617F, we compared the effects of heterozygous, hemizygous and homozygous Jak2V617F expression on hematopoietic signaling. Primary megakaryoblasts and erythroblasts derived from the BM of control (Jak2+/+), heterozygous (Jak2VF/+), hemizygous (Jak2VF/−) and homozygous Jak2V617F (Jak2VF/VF) mice were utilized for signaling studies. Whereas heterozygous Jak2V617F expression resulted in constitutive activation of Stat5, Erk1/2 and/or Akt, hemizygous or homozygous Jak2V617F expression further enhanced the constitutive activation of these signaling pathways (Figures 6a and b). Homozygous Jak2V617F-expressing megakaryoblasts/erythroblasts exhibited the highest level of activation of Stat5, Erk1/2 and Akt signaling (Figures 6a and b). Hemizygous Jak2V617F megakaryoblasts and erythroblasts, which harbored only one allele of Jak2 (Jak2V617F) and thus expressed lower levels of total Jak2 protein than in control, heterozygous or homozygous Jak2V617F megakaryoblasts/erythroblasts, showed greater activation of constitutive signaling compared with control or heterozygous Jak2V617F megakaryoblasts/erythroblasts (Figures 6a and b). Notably, activation of Stat5 and Erk1/2 was much greater in megakaryoblasts than in erythroblasts of hemizygous Jak2V617F mice compared with those in heterozygous Jak2V617F mice (Figures 6a and b). Overall, these results suggest that wild-type Jak2 negatively regulates signaling mediated by Jak2V617F.

Bottom Line: JAK2V617F homozygosity via 9pLOH has been associated with more severe MPN phenotype.Together, these results suggest that loss of Jak2 WT allele increases the severity of the MPN.Thus, the Jak2 WT allele functions as a negative regulator of MPN induced by Jak2V617F.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, USA.

ABSTRACT
JAK2V617F is the most common mutation found in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p (9pLOH) involving the JAK2 locus has been observed in ∼30% of MPN patients. JAK2V617F homozygosity via 9pLOH has been associated with more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the roles of wild-type JAK2 (JAK2 WT) and JAK2V617F alleles in the development of MPNs, we have used conditional Jak2 knock-out and Jak2V617F knock-in mice and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like MPN in mice, loss of Jak2 WT allele in hemizygous or homozygous Jak2V617F mice results in markedly increased white blood cells, neutrophils, reticulocytes and platelets in the peripheral blood, and significantly larger spleen size compared with heterozygous Jak2V617F mice. Hemizygous or homozygous Jak2V617F mice also exhibit accelerated myelofibrosis compared with mice expressing heterozygous Jak2V617F. Together, these results suggest that loss of Jak2 WT allele increases the severity of the MPN. Thus, the Jak2 WT allele functions as a negative regulator of MPN induced by Jak2V617F.

Show MeSH
Related in: MedlinePlus