title: Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.
abstract: Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.
authors.affiliation: Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Ilyas
authors.initials: I
authors.lastname: Chachoua
authors.affiliation: Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Christian
authors.initials: C
authors.lastname: Pecquet
authors.affiliation: INSERM, Unité Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Paris-Saclay, Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Gustave Roussy, Unité Mixte de Recherche 1170, Villejuif, France;
authors.forename: Mira
authors.initials: M
authors.lastname: El-Khoury
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; and.
authors.forename: Harini
authors.initials: H
authors.lastname: Nivarthi
authors.affiliation: Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Roxana-Irina
authors.initials: RI
authors.lastname: Albu
authors.affiliation: INSERM, Unité Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Paris-Saclay, Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Gustave Roussy, Unité Mixte de Recherche 1170, Villejuif, France;
authors.forename: Caroline
authors.initials: C
authors.lastname: Marty
authors.affiliation: Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Vitalina
authors.initials: V
authors.lastname: Gryshkova
authors.affiliation: Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Defour
authors.affiliation: Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Vertenoeil
authors.affiliation: Experimental Therapeutics Center, Singapore.
authors.forename: Anna
authors.initials: A
authors.lastname: Ngo
authors.affiliation: Experimental Therapeutics Center, Singapore.
authors.forename: Ann
authors.initials: A
authors.lastname: Koay
authors.affiliation: INSERM, Unité Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Paris-Saclay, Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Gustave Roussy, Unité Mixte de Recherche 1170, Villejuif, France;
authors.forename: Hana
authors.initials: H
authors.lastname: Raslova
authors.affiliation: Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Courtoy
authors.affiliation: Experimental Therapeutics Center, Singapore.
authors.forename: Meng Ling
authors.initials: ML
authors.lastname: Choong
authors.affiliation: INSERM, Unité Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Paris-Saclay, Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Gustave Roussy, Unité Mixte de Recherche 1170, Villejuif, France;
authors.forename: Isabelle
authors.initials: I
authors.lastname: Plo
authors.affiliation: INSERM, Unité Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Paris-Saclay, Unité Mixte de Recherche 1170, Institut Gustave Roussy, Villejuif, France; Gustave Roussy, Unité Mixte de Recherche 1170, Villejuif, France;
authors.forename: William
authors.initials: W
authors.lastname: Vainchenker
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; and.
authors.forename: Robert
authors.initials: R
authors.lastname: Kralovics
authors.affiliation: Ludwig Institute for Cancer Research Brussels, Brussels, Belgium; Université Catholique de Louvain and de Duve Institute, Brussels, Belgium;
authors.forename: Stefan N
authors.initials: SN
authors.lastname: Constantinescu
mesh_terms: D000818:Animals; D037282:Calreticulin / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D006031:Glycosylation; D019337:Hematologic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism; D060588:LIM Domain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D053628:Receptors, Thrombopoietin / Q000235:genetics / Q000378:metabolism*; D050791:STAT Transcription Factors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D037282:Calreticulin; D053614:Janus Kinase 2; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D009363:Neoplasm Proteins; D019869:Phosphatidylinositol 3-Kinases; D053628:Receptors, Thrombopoietin; D050791:STAT Transcription Factors; C576483:CALR protein, human; C090114:cysteine and glycine-rich protein 3; C082218:MPL protein, human; C507926:Jak2 protein, mouse