Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretionReport as inadecuate




Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion - Download this document for free, or read online. Document in PDF available to download.

Journal of Hematology and Oncology

, 9:45

First Online: 13 May 2016Received: 24 February 2016Accepted: 05 May 2016DOI: 10.1186-s13045-016-0275-0

Cite this article as: Han, L., Schubert, C., Köhler, J. et al. J Hematol Oncol 2016 9: 45. doi:10.1186-s13045-016-0275-0

Abstract

BackgroundSomatic 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.

MethodsThe 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.

ResultsThe 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.

ConclusionsThese 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.

KeywordsCalreticulin Frameshift mutants del52 Myeloproliferative neoplasms MPN Megakaryopoiesis MPL Degradation Protein secretion NF-E2 AbbreviationsBFAbrefeldin A

bpbase pair

Cacalcium

CALRcalreticulin

ERendoplasmic reticulum

ERADER-associated protein degradation

ETessential thrombocythemia

FBSfetal bovine serum

MPNmyeloproliferative neoplasm

MTT3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide

neoneomycin

PMFprimary myelofibrosis

post-ET-MFpost essential thrombocythemia-myelofibrosis

puropuromycin

PVpolycythemia vera

PVDFpolyvinylidene difluoride

RTKreceptor tyrosine kinase

RT-PCRreverse transcription polymerase chain reaction

Ruxoruxolitinib

TPOthrombopoietin

WTwild type

YFPyellow fluorescent protein

Electronic supplementary materialThe online version of this article doi:10.1186-s13045-016-0275-0 contains supplementary material, which is available to authorized users.

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Author: Lijuan Han - Claudia Schubert - Johanna Köhler - Mirle Schemionek - Susanne Isfort - Tim H. Brümmendorf - Steffen Koschm

Source: https://link.springer.com/



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