CALRETICULIN MUTANT PROTEINS INDUCE MEGAKARYOCYTIC SIGNALING TO TRANSFORM HEMATOPOIETIC CELLS AND UNDERGO ACCELERATED DEGRADATION AND GOLGI-MEDIATED SECRETION
(Abstract release date: 05/19/16)
EHA Library. Chatain N. 06/11/16; 135265; S509
Dr. Nicolas Chatain
Contributions
Contributions
Abstract
Abstract: S509
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C13
Background
Somatic calreticulin (CALR), Janus kinase 2 (JAK2) V617F, and thrombopoietin receptor (MPL) mutations are essentially mutually exclusive in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. CALR mutants are confined to MPNs with aberrant megakaryopoiesis, such as essential thrombocythemia and myelofibrosis. Recent data showed that MPL is essential for CALR mutant-driven MPN. However, the characteristics of CALR mutants and their mechanisms of action are still poorly understood.
Aims
Our study was aimed at the elucidation of MPL-dependent and -independent mechanisms of CALR frameshift mutants to drive megakaryocytic differentiation and oncogenic transformation. Furthermore, we investigated the cause of low cellular protein abundance of the CALR mutants.
Methods
The murine myeloid cell line 32D and human HL60 cells stably expressing the most frequent CALR mutants (del52 and ins5) were generated to analyze growth factor-independent growth, as the first steps of cellular transformation, in the presence and absence of MPL expression. Additionally, using brefeldin A, MG132, and tunicamycin treatment as well as generation of further mutants (i.e. YFP-fusion proteins), CALR mutant protein degradation and secretion were examined.
Results
In this report, we demonstrate that the most frequent CALR mutants, type 1 (del52) and type 2 (ins5), increase critical megakaryocytic transcription factors such as NF-E2 and GATA1. Interestingly, this occurred in an MPL-independent but AKT-dependent fashion, leading to the upregulation of Mpl and CD41 expression. These effects were also confirmed by the observation of spontaneous outgrowth of CALR mutant-transduced 32D cells, showing increased NF-E2, CD41, and Mpl expression as well as constitutive STAT5 activation and response to JAK inhibitor treatment. Interestingly, we found high cellular levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Hence, we demonstrate that low CALR mutant protein abundance is a result of accelerated secretion as well as ubiquitin- and proteasome-independent degradation. CALR mutant degradation was attenuated by MPL expression.
Conclusion
Together, our data illustrate how CALR mutants may initiate abnormal megakaryopoiesis, even in the absence of MPL, by a mechanism that most likely involves activated AKT. In addition, our findings show enhanced secretion, MPL-driven protein stabilization, and importance of Golgi signaling of the MPL receptor for CALR-mutant mediated transformation.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Differentiation, Megakaryopoiesis, Mutation, Secretion
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C13
Background
Somatic calreticulin (CALR), Janus kinase 2 (JAK2) V617F, and thrombopoietin receptor (MPL) mutations are essentially mutually exclusive in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. CALR mutants are confined to MPNs with aberrant megakaryopoiesis, such as essential thrombocythemia and myelofibrosis. Recent data showed that MPL is essential for CALR mutant-driven MPN. However, the characteristics of CALR mutants and their mechanisms of action are still poorly understood.
Aims
Our study was aimed at the elucidation of MPL-dependent and -independent mechanisms of CALR frameshift mutants to drive megakaryocytic differentiation and oncogenic transformation. Furthermore, we investigated the cause of low cellular protein abundance of the CALR mutants.
Methods
The murine myeloid cell line 32D and human HL60 cells stably expressing the most frequent CALR mutants (del52 and ins5) were generated to analyze growth factor-independent growth, as the first steps of cellular transformation, in the presence and absence of MPL expression. Additionally, using brefeldin A, MG132, and tunicamycin treatment as well as generation of further mutants (i.e. YFP-fusion proteins), CALR mutant protein degradation and secretion were examined.
Results
In this report, we demonstrate that the most frequent CALR mutants, type 1 (del52) and type 2 (ins5), increase critical megakaryocytic transcription factors such as NF-E2 and GATA1. Interestingly, this occurred in an MPL-independent but AKT-dependent fashion, leading to the upregulation of Mpl and CD41 expression. These effects were also confirmed by the observation of spontaneous outgrowth of CALR mutant-transduced 32D cells, showing increased NF-E2, CD41, and Mpl expression as well as constitutive STAT5 activation and response to JAK inhibitor treatment. Interestingly, we found high cellular levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Hence, we demonstrate that low CALR mutant protein abundance is a result of accelerated secretion as well as ubiquitin- and proteasome-independent degradation. CALR mutant degradation was attenuated by MPL expression.
Conclusion
Together, our data illustrate how CALR mutants may initiate abnormal megakaryopoiesis, even in the absence of MPL, by a mechanism that most likely involves activated AKT. In addition, our findings show enhanced secretion, MPL-driven protein stabilization, and importance of Golgi signaling of the MPL receptor for CALR-mutant mediated transformation.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Differentiation, Megakaryopoiesis, Mutation, Secretion
Abstract: S509
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C13
Background
Somatic calreticulin (CALR), Janus kinase 2 (JAK2) V617F, and thrombopoietin receptor (MPL) mutations are essentially mutually exclusive in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. CALR mutants are confined to MPNs with aberrant megakaryopoiesis, such as essential thrombocythemia and myelofibrosis. Recent data showed that MPL is essential for CALR mutant-driven MPN. However, the characteristics of CALR mutants and their mechanisms of action are still poorly understood.
Aims
Our study was aimed at the elucidation of MPL-dependent and -independent mechanisms of CALR frameshift mutants to drive megakaryocytic differentiation and oncogenic transformation. Furthermore, we investigated the cause of low cellular protein abundance of the CALR mutants.
Methods
The murine myeloid cell line 32D and human HL60 cells stably expressing the most frequent CALR mutants (del52 and ins5) were generated to analyze growth factor-independent growth, as the first steps of cellular transformation, in the presence and absence of MPL expression. Additionally, using brefeldin A, MG132, and tunicamycin treatment as well as generation of further mutants (i.e. YFP-fusion proteins), CALR mutant protein degradation and secretion were examined.
Results
In this report, we demonstrate that the most frequent CALR mutants, type 1 (del52) and type 2 (ins5), increase critical megakaryocytic transcription factors such as NF-E2 and GATA1. Interestingly, this occurred in an MPL-independent but AKT-dependent fashion, leading to the upregulation of Mpl and CD41 expression. These effects were also confirmed by the observation of spontaneous outgrowth of CALR mutant-transduced 32D cells, showing increased NF-E2, CD41, and Mpl expression as well as constitutive STAT5 activation and response to JAK inhibitor treatment. Interestingly, we found high cellular levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Hence, we demonstrate that low CALR mutant protein abundance is a result of accelerated secretion as well as ubiquitin- and proteasome-independent degradation. CALR mutant degradation was attenuated by MPL expression.
Conclusion
Together, our data illustrate how CALR mutants may initiate abnormal megakaryopoiesis, even in the absence of MPL, by a mechanism that most likely involves activated AKT. In addition, our findings show enhanced secretion, MPL-driven protein stabilization, and importance of Golgi signaling of the MPL receptor for CALR-mutant mediated transformation.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Differentiation, Megakaryopoiesis, Mutation, Secretion
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 16:45 - 17:00
Location: Hall C13
Background
Somatic calreticulin (CALR), Janus kinase 2 (JAK2) V617F, and thrombopoietin receptor (MPL) mutations are essentially mutually exclusive in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. CALR mutants are confined to MPNs with aberrant megakaryopoiesis, such as essential thrombocythemia and myelofibrosis. Recent data showed that MPL is essential for CALR mutant-driven MPN. However, the characteristics of CALR mutants and their mechanisms of action are still poorly understood.
Aims
Our study was aimed at the elucidation of MPL-dependent and -independent mechanisms of CALR frameshift mutants to drive megakaryocytic differentiation and oncogenic transformation. Furthermore, we investigated the cause of low cellular protein abundance of the CALR mutants.
Methods
The murine myeloid cell line 32D and human HL60 cells stably expressing the most frequent CALR mutants (del52 and ins5) were generated to analyze growth factor-independent growth, as the first steps of cellular transformation, in the presence and absence of MPL expression. Additionally, using brefeldin A, MG132, and tunicamycin treatment as well as generation of further mutants (i.e. YFP-fusion proteins), CALR mutant protein degradation and secretion were examined.
Results
In this report, we demonstrate that the most frequent CALR mutants, type 1 (del52) and type 2 (ins5), increase critical megakaryocytic transcription factors such as NF-E2 and GATA1. Interestingly, this occurred in an MPL-independent but AKT-dependent fashion, leading to the upregulation of Mpl and CD41 expression. These effects were also confirmed by the observation of spontaneous outgrowth of CALR mutant-transduced 32D cells, showing increased NF-E2, CD41, and Mpl expression as well as constitutive STAT5 activation and response to JAK inhibitor treatment. Interestingly, we found high cellular levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Hence, we demonstrate that low CALR mutant protein abundance is a result of accelerated secretion as well as ubiquitin- and proteasome-independent degradation. CALR mutant degradation was attenuated by MPL expression.
Conclusion
Together, our data illustrate how CALR mutants may initiate abnormal megakaryopoiesis, even in the absence of MPL, by a mechanism that most likely involves activated AKT. In addition, our findings show enhanced secretion, MPL-driven protein stabilization, and importance of Golgi signaling of the MPL receptor for CALR-mutant mediated transformation.
Session topic: Myeloproliferative neoplasms - Biology
Keyword(s): Differentiation, Megakaryopoiesis, Mutation, Secretion
{{ help_message }}
{{filter}}