Chronic myeloproliferative neoplasms

Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development

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Abstract

Frameshifting mutations (−1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.

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Acknowledgements

We thank Dr Patrick Jacquemin for advice on CRISPR/Cas9 and transgenesis, Dr Pedro Gomez for support with mouse facility, Dr Nicolas Dauguet for expert flow cytometry support, Dr Jing Jing Zhu for her help with the T-cell intracellular staining assay, Lidvine Genet and Céline Mouton for expert technical and administrative support. TB was supported by a Télévie PhD Fellowship. SNC is Honorary Research Director at FRS-FNRS Belgium. Funding to SNC is acknowledged from Ludwig Institute for Cancer Research, Fondation contre le cancer, Fondation « Les avions de Sébastien », Fondation Salus Sanguinis and projects Action de Recherche Concertée (ARC) 16/21-073 and WelBio (Walloon Excellence In Life Sciences and Biotechnology F 44/8/5- MCF/UIG-10955), Brussels, Belgium.

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Correspondence to Stefan N. Constantinescu.

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RK and SNC are cofounders of MyeloPro Research and Diagnostics GmbH, Vienna, Austria. The remaining authors declare that they have no conflict of interest.

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