title: Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion.
abstract: Somatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated.
  
  
  
  The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment.
  
  
  
  The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation.
  
  
  
  These findings demonstrate the potency of CALR mutants to drive expression of megakaryocytic differentiation markers such as NF-E2 and CD41 as well as Mpl. Furthermore, CALR mutants undergo accelerated protein degradation that involves the secretory pathway and/or protein glycosylation.
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Han
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Schubert
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Köhler
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Mirle
authors.initials: M
authors.lastname: Schemionek
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Isfort
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Tim H
authors.initials: TH
authors.lastname: Brümmendorf
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany. skoschmieder@ukaachen.de.
authors.forename: Steffen
authors.initials: S
authors.lastname: Koschmieder
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Chatain
mesh_terms: D000818:Animals; D037282:Calreticulin / Q000235:genetics* / Q000502:physiology; D016923:Cell Death; D002460:Cell Line; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D016368:Frameshift Mutation; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000502:physiology*; D009196:Myeloproliferative Disorders / Q000235:genetics; D050984:NF-E2 Transcription Factor, p45 Subunit / Q000032:analysis; D040201:Platelet Membrane Glycoprotein IIb / Q000032:analysis; D059748:Proteolysis; D053628:Receptors, Thrombopoietin / Q000032:analysis; D015398:Signal Transduction
chemical_list: C576483:CALR protein, human; D037282:Calreticulin; D050505:Mutant Proteins; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D040201:Platelet Membrane Glycoprotein IIb; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human
keywords: Calreticulin; Degradation; Frameshift mutants; MPL; MPN; Megakaryopoiesis; Myeloproliferative neoplasms; NF-E2; Protein secretion; del52