TUMOR NECROSIS FACTOR RECEPTOR 2 MAY BE REQUIRED FOR CLONAL DOMINANCE OF JAK2 V617F MUTANT OVER JAK2 WILDTYPE CELLS
(Abstract release date: 05/19/16)
EHA Library. W Deininger M. 06/11/16; 135263; S507
Prof. Michael W Deininger
Contributions
Contributions
Abstract
Abstract: S507
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:15 - 16:30
Location: Hall C13
Background
The JAK2V617F mutation is detected in large subsets of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN, including tumor necrosis factor alpha (TNF-α). We have previously shown that JAK2 kinase activity regulates TNF-α expression, which in turn imparts a competitive advantage on JAK2V617F expressing cells over their normal counterparts (Fleischman et al. Blood. 2011;118(24):6392-8).
Aims
1. To identify the cellular source of TNF-α generation in JAK2V617 expressing hematopoietic cells.
2. To deliniate whether TNFR1 vs TNFR2 mediates TNF effects in MPNs.
Methods
We used FACS/intracellular staining to measure TNF-α expression in murine and human hematopoietic cells. Blocking antibodies or shRNA knockdown were used to specifically probe the role of TNFR1 vs TNFR2 for MPN colony formation. Colonies were genotyped by FACS (GFP) or DNA sequencing. TNFR1/R2 null hematopoietic cells were used in competitive repopulation experiments to interogate the functional relevance of TNFR1 vs TNFR2 for MPN pathogenesis.
Results
TNF-α was low in unstimulated hematopoietic cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), with significant but less pronounced differences in more mature populations. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing hematopoietic stem cells (HSCs; GFP+) compared to GFP- controls (p<0.005), irrespective of LPS stimulation. We initially treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α, but found that etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of plasma TNF-α activity. As TNF-α may activate pro-apoptotic (predominantly through TNFR1) and pro-survival pathways (predominantly through TNFR2), we reasoned that global (etanercept) blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Next we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2 and assessed colony formation. Blocking TNFR2 resulted in decreased colony formation, while blocking TNFR1 increased colony formation. Analogous results were seen in CD34+ cells from MF patients. Additionally in samples heterozygous for JAK2V617F, TNFR2-blocking antibodies selected JAK2 wild type over V617F mutant colonies. Preliminary results from murine TNFR1 and TNFR2 null models and shRNA knockdown of TNFR1 and TNFR2 suggest that lack of TNFR2 but not TNFR1 favors outgrowth of wild type cells.
Conclusion
Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, suggesting that selective inhibition of TNFR2 may shift the equilibrium from MPN toward normal hematopoiesis and that TNFR2 blockage may be useful to treat MF and other myeloproliferative neoplasms.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Cytokine, Myelofibrosis, Myeloproliferative disorder
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:15 - 16:30
Location: Hall C13
Background
The JAK2V617F mutation is detected in large subsets of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN, including tumor necrosis factor alpha (TNF-α). We have previously shown that JAK2 kinase activity regulates TNF-α expression, which in turn imparts a competitive advantage on JAK2V617F expressing cells over their normal counterparts (Fleischman et al. Blood. 2011;118(24):6392-8).
Aims
1. To identify the cellular source of TNF-α generation in JAK2V617 expressing hematopoietic cells.
2. To deliniate whether TNFR1 vs TNFR2 mediates TNF effects in MPNs.
Methods
We used FACS/intracellular staining to measure TNF-α expression in murine and human hematopoietic cells. Blocking antibodies or shRNA knockdown were used to specifically probe the role of TNFR1 vs TNFR2 for MPN colony formation. Colonies were genotyped by FACS (GFP) or DNA sequencing. TNFR1/R2 null hematopoietic cells were used in competitive repopulation experiments to interogate the functional relevance of TNFR1 vs TNFR2 for MPN pathogenesis.
Results
TNF-α was low in unstimulated hematopoietic cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), with significant but less pronounced differences in more mature populations. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing hematopoietic stem cells (HSCs; GFP+) compared to GFP- controls (p<0.005), irrespective of LPS stimulation. We initially treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α, but found that etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of plasma TNF-α activity. As TNF-α may activate pro-apoptotic (predominantly through TNFR1) and pro-survival pathways (predominantly through TNFR2), we reasoned that global (etanercept) blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Next we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2 and assessed colony formation. Blocking TNFR2 resulted in decreased colony formation, while blocking TNFR1 increased colony formation. Analogous results were seen in CD34+ cells from MF patients. Additionally in samples heterozygous for JAK2V617F, TNFR2-blocking antibodies selected JAK2 wild type over V617F mutant colonies. Preliminary results from murine TNFR1 and TNFR2 null models and shRNA knockdown of TNFR1 and TNFR2 suggest that lack of TNFR2 but not TNFR1 favors outgrowth of wild type cells.
Conclusion
Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, suggesting that selective inhibition of TNFR2 may shift the equilibrium from MPN toward normal hematopoiesis and that TNFR2 blockage may be useful to treat MF and other myeloproliferative neoplasms.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Cytokine, Myelofibrosis, Myeloproliferative disorder
Abstract: S507
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:15 - 16:30
Location: Hall C13
Background
The JAK2V617F mutation is detected in large subsets of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN, including tumor necrosis factor alpha (TNF-α). We have previously shown that JAK2 kinase activity regulates TNF-α expression, which in turn imparts a competitive advantage on JAK2V617F expressing cells over their normal counterparts (Fleischman et al. Blood. 2011;118(24):6392-8).
Aims
1. To identify the cellular source of TNF-α generation in JAK2V617 expressing hematopoietic cells.
2. To deliniate whether TNFR1 vs TNFR2 mediates TNF effects in MPNs.
Methods
We used FACS/intracellular staining to measure TNF-α expression in murine and human hematopoietic cells. Blocking antibodies or shRNA knockdown were used to specifically probe the role of TNFR1 vs TNFR2 for MPN colony formation. Colonies were genotyped by FACS (GFP) or DNA sequencing. TNFR1/R2 null hematopoietic cells were used in competitive repopulation experiments to interogate the functional relevance of TNFR1 vs TNFR2 for MPN pathogenesis.
Results
TNF-α was low in unstimulated hematopoietic cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), with significant but less pronounced differences in more mature populations. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing hematopoietic stem cells (HSCs; GFP+) compared to GFP- controls (p<0.005), irrespective of LPS stimulation. We initially treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α, but found that etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of plasma TNF-α activity. As TNF-α may activate pro-apoptotic (predominantly through TNFR1) and pro-survival pathways (predominantly through TNFR2), we reasoned that global (etanercept) blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Next we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2 and assessed colony formation. Blocking TNFR2 resulted in decreased colony formation, while blocking TNFR1 increased colony formation. Analogous results were seen in CD34+ cells from MF patients. Additionally in samples heterozygous for JAK2V617F, TNFR2-blocking antibodies selected JAK2 wild type over V617F mutant colonies. Preliminary results from murine TNFR1 and TNFR2 null models and shRNA knockdown of TNFR1 and TNFR2 suggest that lack of TNFR2 but not TNFR1 favors outgrowth of wild type cells.
Conclusion
Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, suggesting that selective inhibition of TNFR2 may shift the equilibrium from MPN toward normal hematopoiesis and that TNFR2 blockage may be useful to treat MF and other myeloproliferative neoplasms.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Cytokine, Myelofibrosis, Myeloproliferative disorder
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:15 - 16:30
Location: Hall C13
Background
The JAK2V617F mutation is detected in large subsets of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN, including tumor necrosis factor alpha (TNF-α). We have previously shown that JAK2 kinase activity regulates TNF-α expression, which in turn imparts a competitive advantage on JAK2V617F expressing cells over their normal counterparts (Fleischman et al. Blood. 2011;118(24):6392-8).
Aims
1. To identify the cellular source of TNF-α generation in JAK2V617 expressing hematopoietic cells.
2. To deliniate whether TNFR1 vs TNFR2 mediates TNF effects in MPNs.
Methods
We used FACS/intracellular staining to measure TNF-α expression in murine and human hematopoietic cells. Blocking antibodies or shRNA knockdown were used to specifically probe the role of TNFR1 vs TNFR2 for MPN colony formation. Colonies were genotyped by FACS (GFP) or DNA sequencing. TNFR1/R2 null hematopoietic cells were used in competitive repopulation experiments to interogate the functional relevance of TNFR1 vs TNFR2 for MPN pathogenesis.
Results
TNF-α was low in unstimulated hematopoietic cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), with significant but less pronounced differences in more mature populations. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing hematopoietic stem cells (HSCs; GFP+) compared to GFP- controls (p<0.005), irrespective of LPS stimulation. We initially treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α, but found that etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of plasma TNF-α activity. As TNF-α may activate pro-apoptotic (predominantly through TNFR1) and pro-survival pathways (predominantly through TNFR2), we reasoned that global (etanercept) blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Next we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2 and assessed colony formation. Blocking TNFR2 resulted in decreased colony formation, while blocking TNFR1 increased colony formation. Analogous results were seen in CD34+ cells from MF patients. Additionally in samples heterozygous for JAK2V617F, TNFR2-blocking antibodies selected JAK2 wild type over V617F mutant colonies. Preliminary results from murine TNFR1 and TNFR2 null models and shRNA knockdown of TNFR1 and TNFR2 suggest that lack of TNFR2 but not TNFR1 favors outgrowth of wild type cells.
Conclusion
Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, suggesting that selective inhibition of TNFR2 may shift the equilibrium from MPN toward normal hematopoiesis and that TNFR2 blockage may be useful to treat MF and other myeloproliferative neoplasms.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Cytokine, Myelofibrosis, Myeloproliferative disorder
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