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: Ilyas Chachoua, Christian Pecquet, Mira El-Khoury, Harini Nivarthi, Roxana-Irina Albu, Caroline Marty, Vitalina Gryshkova, Jean-Philippe Defour, Gaëlle Vertenoeil, Anna Ngo, Ann Koay, Hana Raslova, Pierre J Courtoy, Meng Ling Choong, Isabelle Plo, William Vainchenker, Robert Kralovics, Stefan N Constantinescu
• Journal: Blood (Blood) Vol. 127 Issue 10 Pg. 1325-35 (Mar 10 2016) ISSN: 1528-0020 [Electronic] United States
• PMID: 26668133 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2016 by The American Society of Hematology.

Chemical References

• CALR protein, human
• Calreticulin
• LIM Domain Proteins
• Muscle Proteins
• Neoplasm Proteins
• Receptors, Thrombopoietin
• STAT Transcription Factors
• cysteine and glycine-rich protein 3
• MPL protein, human
• Jak2 protein, mouse
• Janus Kinase 2

Topics

• Animals
• Calreticulin (genetics, metabolism)
• Cell Line, Tumor
• Glycosylation
• Hematologic Neoplasms (genetics, metabolism, pathology)
• Janus Kinase 2 (genetics, metabolism)
• LIM Domain Proteins (genetics, metabolism)
• Mice
• Muscle Proteins (genetics, metabolism)
• Mutation
• Myeloproliferative Disorders (genetics, metabolism, pathology)
• Neoplasm Proteins (genetics, metabolism)
• Phosphatidylinositol 3-Kinases (genetics, metabolism)
• Phosphorylation (genetics)
• Protein Transport (genetics)
• Receptors, Thrombopoietin (genetics, metabolism)
• STAT Transcription Factors (genetics, metabolism)
• Signal Transduction (genetics)