ACTIVATION OF THE THROMBOPOIETIN RECEPTOR BY MUTANT CALRETICULIN IN CALR-MUTANT MYELOPROLIFERATIVE NEOPLASMS
(Abstract release date: 05/19/16)
EHA Library. Araki M. 06/11/16; 135212; S456
Dr. Marito Araki
Contributions
Contributions
Abstract
Abstract: S456
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:30 - 12:45
Location: Hall A3
Background
Recurrent somatic mutations of Calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains to be elucidated.
Aims
We aim to elucidate the oncogenic property of mutant CALR and molecular mechanism of its action.
Methods
To examine whether mutant CALR exhibits oncogenic properties, its capacity to induce factor-independent growth was examined in a human megakaryocytic cell line UT-7/TPO that proliferates only in the presence of thrombopoietin (TPO). The genetic and biochemical interactions between mutant CALR and TPO receptor c-MPL were examined using a loss-of-function assay with shRNA and a co-immunoprecipitation assay, respectively. The functional and structural differences between mutant and wild-type CALR were examined using a domain analysis of CALRs for the c-MPL interaction. Mutant-CALR activation of c-MPL was determined by examining the phosphorylation status of c-MPL-downstream molecules using an immunoblot analysis. To demonstrate this activation process in more physiological circumstances, a loss-of-function assay for c-MPL was performed in iPS-derived hematopoietic stem cells that harbor the CALR mutation and exhibit TPO-independent megakaryopoiesis.
Results
We found that the expression of mutant, but not wild-type, CALR induced TPO-independent growth of UT-7/TPO cells. We demonstrated that c-MPL was required for this cytokine-independent growth. Mutant CALR preferentially associated with c-MPL that is bound to JAK2 over the wild-type protein. Furthermore, we demonstrated that the mutant-specific C-terminus portion of CALR interfered with the P-domain of CALR to allow the N-domain to interact with c-MPL; thus, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induced the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked when the cells were treated with a JAK2 inhibitor. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in iPS-derived hematopoietic stem cells harboring the CALR mutation.
Conclusion
These findings imply that mutant CALR activated the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes the development of myeloproliferative neoplasms by activating c-MPL and its downstream pathway.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): C-mpl, TPO
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:30 - 12:45
Location: Hall A3
Background
Recurrent somatic mutations of Calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains to be elucidated.
Aims
We aim to elucidate the oncogenic property of mutant CALR and molecular mechanism of its action.
Methods
To examine whether mutant CALR exhibits oncogenic properties, its capacity to induce factor-independent growth was examined in a human megakaryocytic cell line UT-7/TPO that proliferates only in the presence of thrombopoietin (TPO). The genetic and biochemical interactions between mutant CALR and TPO receptor c-MPL were examined using a loss-of-function assay with shRNA and a co-immunoprecipitation assay, respectively. The functional and structural differences between mutant and wild-type CALR were examined using a domain analysis of CALRs for the c-MPL interaction. Mutant-CALR activation of c-MPL was determined by examining the phosphorylation status of c-MPL-downstream molecules using an immunoblot analysis. To demonstrate this activation process in more physiological circumstances, a loss-of-function assay for c-MPL was performed in iPS-derived hematopoietic stem cells that harbor the CALR mutation and exhibit TPO-independent megakaryopoiesis.
Results
We found that the expression of mutant, but not wild-type, CALR induced TPO-independent growth of UT-7/TPO cells. We demonstrated that c-MPL was required for this cytokine-independent growth. Mutant CALR preferentially associated with c-MPL that is bound to JAK2 over the wild-type protein. Furthermore, we demonstrated that the mutant-specific C-terminus portion of CALR interfered with the P-domain of CALR to allow the N-domain to interact with c-MPL; thus, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induced the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked when the cells were treated with a JAK2 inhibitor. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in iPS-derived hematopoietic stem cells harboring the CALR mutation.
Conclusion
These findings imply that mutant CALR activated the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes the development of myeloproliferative neoplasms by activating c-MPL and its downstream pathway.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): C-mpl, TPO
Abstract: S456
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:30 - 12:45
Location: Hall A3
Background
Recurrent somatic mutations of Calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains to be elucidated.
Aims
We aim to elucidate the oncogenic property of mutant CALR and molecular mechanism of its action.
Methods
To examine whether mutant CALR exhibits oncogenic properties, its capacity to induce factor-independent growth was examined in a human megakaryocytic cell line UT-7/TPO that proliferates only in the presence of thrombopoietin (TPO). The genetic and biochemical interactions between mutant CALR and TPO receptor c-MPL were examined using a loss-of-function assay with shRNA and a co-immunoprecipitation assay, respectively. The functional and structural differences between mutant and wild-type CALR were examined using a domain analysis of CALRs for the c-MPL interaction. Mutant-CALR activation of c-MPL was determined by examining the phosphorylation status of c-MPL-downstream molecules using an immunoblot analysis. To demonstrate this activation process in more physiological circumstances, a loss-of-function assay for c-MPL was performed in iPS-derived hematopoietic stem cells that harbor the CALR mutation and exhibit TPO-independent megakaryopoiesis.
Results
We found that the expression of mutant, but not wild-type, CALR induced TPO-independent growth of UT-7/TPO cells. We demonstrated that c-MPL was required for this cytokine-independent growth. Mutant CALR preferentially associated with c-MPL that is bound to JAK2 over the wild-type protein. Furthermore, we demonstrated that the mutant-specific C-terminus portion of CALR interfered with the P-domain of CALR to allow the N-domain to interact with c-MPL; thus, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induced the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked when the cells were treated with a JAK2 inhibitor. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in iPS-derived hematopoietic stem cells harboring the CALR mutation.
Conclusion
These findings imply that mutant CALR activated the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes the development of myeloproliferative neoplasms by activating c-MPL and its downstream pathway.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): C-mpl, TPO
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:30 - 12:45
Location: Hall A3
Background
Recurrent somatic mutations of Calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains to be elucidated.
Aims
We aim to elucidate the oncogenic property of mutant CALR and molecular mechanism of its action.
Methods
To examine whether mutant CALR exhibits oncogenic properties, its capacity to induce factor-independent growth was examined in a human megakaryocytic cell line UT-7/TPO that proliferates only in the presence of thrombopoietin (TPO). The genetic and biochemical interactions between mutant CALR and TPO receptor c-MPL were examined using a loss-of-function assay with shRNA and a co-immunoprecipitation assay, respectively. The functional and structural differences between mutant and wild-type CALR were examined using a domain analysis of CALRs for the c-MPL interaction. Mutant-CALR activation of c-MPL was determined by examining the phosphorylation status of c-MPL-downstream molecules using an immunoblot analysis. To demonstrate this activation process in more physiological circumstances, a loss-of-function assay for c-MPL was performed in iPS-derived hematopoietic stem cells that harbor the CALR mutation and exhibit TPO-independent megakaryopoiesis.
Results
We found that the expression of mutant, but not wild-type, CALR induced TPO-independent growth of UT-7/TPO cells. We demonstrated that c-MPL was required for this cytokine-independent growth. Mutant CALR preferentially associated with c-MPL that is bound to JAK2 over the wild-type protein. Furthermore, we demonstrated that the mutant-specific C-terminus portion of CALR interfered with the P-domain of CALR to allow the N-domain to interact with c-MPL; thus, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induced the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked when the cells were treated with a JAK2 inhibitor. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in iPS-derived hematopoietic stem cells harboring the CALR mutation.
Conclusion
These findings imply that mutant CALR activated the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes the development of myeloproliferative neoplasms by activating c-MPL and its downstream pathway.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): C-mpl, TPO
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