TY - JOUR
T1 - Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants
AU - Chachoua, Ilyas
AU - Pecquet, Christian
AU - El-Khoury, Mira
AU - Nivarthi, Harini
AU - Albu, Roxana Irina
AU - Marty, Caroline
AU - Gryshkova, Vitalina
AU - Defour, Jean Philippe
AU - Vertenoeil, Gäelle
AU - Ngo, Anna
AU - Koay, Ann
AU - Raslova, Hana
AU - Courtoy, Pierre J.
AU - Choong, Meng Ling
AU - Plo, Isabelle
AU - Vainchenker, William
AU - Kralovics, Robert
AU - Constantinescu, Stefan N.
N1 - Publisher Copyright:
© 2016 by The American Society of Hematology.
PY - 2016/3/10
Y1 - 2016/3/10
N2 - Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a 21/12 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-39-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 cytokineindependent 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.
AB - Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a 21/12 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-39-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 cytokineindependent 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.
UR - http://www.scopus.com/inward/record.url?scp=84960851640&partnerID=8YFLogxK
U2 - 10.1182/blood-2015-11-681932
DO - 10.1182/blood-2015-11-681932
M3 - Article
C2 - 26668133
AN - SCOPUS:84960851640
SN - 0006-4971
VL - 127
SP - 1325
EP - 1335
JO - Blood
JF - Blood
IS - 10
ER -