title: Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism.
abstract: Mutations in the calreticulin gene (CALR) were recently identified in approximately 70-80% of patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C-terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium-binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL-3-independent growth. Interestingly, expression of type I and type II mutant CALR in a nonhematopoietic cell line does not directly activate JAK/STAT signaling compared to wildtype CALR and JAK2-V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll-like receptor agonist. These effects are not dependent on the novel C-terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis.
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Garbati
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Welgan
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Sally H
authors.initials: SH
authors.lastname: Landefeld
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Laura F
authors.initials: LF
authors.lastname: Newell
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Anupriya
authors.initials: A
authors.lastname: Agarwal
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.|Department of Pathology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Dunlap
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Chourasia
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Hyunjung
authors.initials: H
authors.lastname: Lee
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Johannes
authors.initials: J
authors.lastname: Elferich
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Elie
authors.initials: E
authors.lastname: Traer
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.|Department of Pathology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Rogan
authors.initials: R
authors.lastname: Rattray
authors.affiliation: Department of Pathology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Cascio
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.|Department of Pathology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Press
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Grover C
authors.initials: GC
authors.lastname: Bagby
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.|Department of Cell, Development, and Cancer Biology, Oregon Health and Science University, Knight Cancer Institute, Portland, Oregon.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Tyner
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.|Department of Cell, Development, and Cancer Biology, Oregon Health and Science University, Knight Cancer Institute, Portland, Oregon.|Howard Hughes Medical Institute, Oregon Health and Science University, Portland, Oregon.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Druker
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Kim-Hien T
authors.initials: KH
authors.lastname: Dao
mesh_terms: D015153:Blotting, Western; D001853:Bone Marrow / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D037282:Calreticulin / Q000235:genetics* / Q000378:metabolism; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000378:metabolism; D017077:Culture Media, Conditioned; D016207:Cytokines / Q000096:biosynthesis; D005110:Extracellular Space / Q000378:metabolism; D016368:Frameshift Mutation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D053614:Janus Kinase 2 / Q000235:genetics; D009000:Monocytes / Q000378:metabolism* / Q000502:physiology; D019899:Paracrine Communication / Q000235:genetics*; D055728:Primary Myelofibrosis / Q000235:genetics / Q000276:immunology; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction; D013920:Thrombocythemia, Essential / Q000235:genetics / Q000276:immunology
chemical_list: C576483:CALR protein, human; D037282:Calreticulin; D017077:Culture Media, Conditioned; D016207:Cytokines; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D002118:Calcium