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

Overview

Journal Leukemia

Specialties Hematology
Oncology

Date 2019 Sep 1

PMID 31471561

Citations 21

Authors

Thomas Balligand

Younes Achouri

Christian Pecquet

Gilles Gaudray

Didier Colau

Eva Hug

Yacine Rahmani

Vincent Stroobant

Isabelle Plo

William Vainchenker

Robert Kralovics

Benoit J Van den Eynde

Jean-Philippe Defour

Stefan N Constantinescu

Affiliations

Soon will be listed here.

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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References

Arber D, Orazi A, Hasserjian R, Thiele J, Borowitz M, Le Beau M . The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016; 127(20):2391-405. DOI: 10.1182/blood-2016-03-643544. View

Kralovics R, Passamonti F, Buser A, Teo S, Tiedt R, Passweg J . A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352(17):1779-90. DOI: 10.1056/NEJMoa051113. View

James C, Ugo V, Le Couedic J, Staerk J, Delhommeau F, Lacout C . A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005; 434(7037):1144-8. DOI: 10.1038/nature03546. View

Levine R, Wadleigh M, Cools J, Ebert B, Wernig G, Huntly B . Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005; 7(4):387-97. DOI: 10.1016/j.ccr.2005.03.023. View

Baxter E, Scott L, Campbell P, East C, Fourouclas N, Swanton S . Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005; 365(9464):1054-61. DOI: 10.1016/S0140-6736(05)71142-9. View

Klampfl T, Gisslinger H, Harutyunyan A, Nivarthi H, Rumi E, Milosevic J . Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013; 369(25):2379-90. DOI: 10.1056/NEJMoa1311347. View

Nangalia J, Massie C, Baxter E, Nice F, Gundem G, Wedge D . Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013; 369(25):2391-2405. PMC: 3966280. DOI: 10.1056/NEJMoa1312542. View

Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu R, Marty C . Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. Blood. 2015; 127(10):1325-35. DOI: 10.1182/blood-2015-11-681932. View

Araki M, Yang Y, Masubuchi N, Hironaka Y, Takei H, Morishita S . Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms. Blood. 2016; 127(10):1307-16. DOI: 10.1182/blood-2015-09-671172. View

10.

Elf S, Abdelfattah N, Chen E, Perales-Paton J, Rosen E, Ko A . Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation. Cancer Discov. 2016; 6(4):368-81. PMC: 4851866. DOI: 10.1158/2159-8290.CD-15-1434. View

11.

Elf S, Abdelfattah N, Baral A, Beeson D, Rivera J, Ko A . Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN. Blood. 2017; 131(7):782-786. PMC: 5814933. DOI: 10.1182/blood-2017-08-800896. View

12.

Marty C, Pecquet C, Nivarthi H, El-Khoury M, Chachoua I, Tulliez M . Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. Blood. 2015; 127(10):1317-24. DOI: 10.1182/blood-2015-11-679571. View

13.

Nivarthi H, Chen D, Cleary C, Kubesova B, Jager R, Bogner E . Thrombopoietin receptor is required for the oncogenic function of CALR mutants. Leukemia. 2016; 30(8):1759-63. PMC: 4980558. DOI: 10.1038/leu.2016.32. View

14.

Han L, Schubert C, Kohler J, Schemionek M, Isfort S, Brummendorf T . Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion. J Hematol Oncol. 2016; 9(1):45. PMC: 4894373. DOI: 10.1186/s13045-016-0275-0. View

15.

Pecquet C, Chachoua I, Roy A, Balligand T, Vertenoeil G, Leroy E . Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants. Blood. 2019; 133(25):2669-2681. DOI: 10.1182/blood-2018-09-874578. View

16.

Balligand T, Achouri Y, Pecquet C, Chachoua I, Nivarthi H, Marty C . Pathologic activation of thrombopoietin receptor and JAK2-STAT5 pathway by frameshift mutants of mouse calreticulin. Leukemia. 2016; 30(8):1775-8. DOI: 10.1038/leu.2016.47. View

17.

Shide K, Kameda T, Yamaji T, Sekine M, Inada N, Kamiunten A . Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib. Leukemia. 2016; 31(5):1136-1144. PMC: 5420793. DOI: 10.1038/leu.2016.308. View

18.

Li J, Prins D, Park H, Grinfeld J, Gonzalez-Arias C, Loughran S . Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage. Blood. 2017; 131(6):649-661. DOI: 10.1182/blood-2017-09-806356. View

19.

Shide K, Kameda T, Kamiunten A, Oji A, Ozono Y, Sekine M . Mice with Calr mutations homologous to human CALR mutations only exhibit mild thrombocytosis. Blood Cancer J. 2019; 9(4):42. PMC: 6440999. DOI: 10.1038/s41408-019-0202-z. View

20.

Cong L, Ran F, Cox D, Lin S, Barretto R, Habib N . Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-23. PMC: 3795411. DOI: 10.1126/science.1231143. View