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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

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

  1. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al. The2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127:2391.

    CAS  PubMed  Article  Google Scholar 

  2. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352:1779–90.

    CAS  PubMed  Article  Google Scholar 

  3. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434:1144–8.

    CAS  PubMed  Article  Google Scholar 

  4. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005;7:387–97.

    CAS  PubMed  Article  Google Scholar 

  5. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365:1054–61.

    CAS  PubMed  Article  Google Scholar 

  6. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369:2379–90.

    CAS  PubMed  Article  Google Scholar 

  7. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369:2391–405.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu RI, Marty C, et al. Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. Blood. 2016;127:1325–35.

    CAS  PubMed  Article  Google Scholar 

  9. Araki M, Yang Y, Masubuchi N, Hironaka Y, Takei H, Morishita S, et al. Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms. Blood. 2016;127:1307–16.

    CAS  PubMed  Article  Google Scholar 

  10. Elf S, Abdelfattah NS, Chen E, Perales-Paton J, Rosen EA, Ko A, et al. Mutant calreticulin requires both its mutant C-terminus and the thrombopoietin receptor for oncogenic transformation. Cancer Discov. 2016;6:368–81.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Elf S, Abdelfattah NS, Baral AJ, Beeson D, Rivera JF, Ko A, et al. Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN. Blood. 2018;131:782–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Marty C, Pecquet C, Nivarthi H, El-Khoury M, Chachoua I, Tulliez M, et al. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. Blood. 2016;127:1317–24.

    CAS  PubMed  Article  Google Scholar 

  13. Nivarthi H, Chen D, Cleary C, Kubesova B, Jager R, Bogner E, et al. Thrombopoietin receptor is required for the oncogenic function of CALR mutants. Leukemia. 2016;30:1759–63.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Han L, Schubert C, Kohler J, Schemionek M, Isfort S, Brummendorf TH, et al. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion. J Hematol Oncol. 2016;9:45.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  15. Pecquet C, Chachoua I, Roy A, Balligand T, Vertenoeil G, Leroy E, et al. Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants. Blood. 2019;133:2669–81.

    CAS  PubMed  Article  Google Scholar 

  16. Balligand T, Achouri Y, Pecquet C, Chachoua I, Nivarthi H, Marty C, et al. Pathologic activation of thrombopoietin receptor and JAK2-STAT5 pathway by frameshift mutants of mouse calreticulin. Leukemia. 2016;30:1775–8.

    CAS  PubMed  Article  Google Scholar 

  17. Shide K, Kameda T, Yamaji T, Sekine M, Inada N, Kamiunten A, et al. Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib. Leukemia. 2016;31:1136–44.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  18. Li J, Prins D, Park HJ, Grinfeld J, Gonzalez-Arias C, Loughran S, et al. Mutant calreticulin knock-in mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage. Blood. 2018;131:649–61.

    CAS  PubMed  Article  Google Scholar 

  19. Shide K, Kameda T, Kamiunten A, Oji A, Ozono Y, Sekine M, et al. Mice with Calr mutations homologous to human CALR mutations only exhibit mild thrombocytosis. Blood Cancer J. 2019;9:42.

    PubMed  PubMed Central  Article  Google Scholar 

  20. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep. 2013;3:3355.

    PubMed  PubMed Central  Article  Google Scholar 

  22. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337:816–21.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Balligand T, Achouri Y, Chachoua I, Pecquet C, Defour J-P, Constantinescu SN. Crispr/Cas9 engineered 61bp deletion in the Calr gene of mice leads to development of thrombocytosis. Blood. 2016;128:4274-.

    Article  Google Scholar 

  24. Kollmann K, Nangalia J, Warsch W, Quentmeier H, Bench A, Boyd E, et al. MARIMO cells harbor a CALR mutation but are not dependent on JAK2/STAT5 signaling. Leukemia. 2015;29:494–7.

    CAS  PubMed  Article  Google Scholar 

  25. Yoshida H, Kondo M, Ichihashi T, Hashimoto N, Inazawa J, Ohno R, et al. A novel myeloid cell line, Marimo, derived from therapy-related acute myeloid leukemia during treatment of essential thrombocythemia: consistent chromosomal abnormalities and temporary C-MYC gene amplification. Cancer Genet Cytogenet. 1998;100:21–4.

    CAS  PubMed  Article  Google Scholar 

  26. Han L, Czech J, Maurer A, Brummendorf TH, Chatain N, Koschmieder S. Mutant NRAS Q61K is responsible for MAPK pathway activation in the MARIMO cell line and renders these cells independent of the CALR-MPL-JAK2-STAT5 pathway. Leukemia. 2018;32:2087–90.

    CAS  PubMed  Article  Google Scholar 

  27. Staerk J, Defour JP, Pecquet C, Leroy E, Antoine-Poirel H, Brett I, et al. Orientation-specific signalling by thrombopoietin receptor dimers. Embo J. 2011;30:4398–413.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Defour JP, Itaya M, Gryshkova V, Brett IC, Pecquet C, Sato T, et al. Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation. Proc Natl Acad Sci USA. 2013;110:2540–5.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. Leroy E, Defour JP, Sato T, Dass S, Gryshkova V, Shwe MM, et al. His499 regulates dimerization and prevents oncogenic activation by asparagine mutations of the human thrombopoietin receptor. J Biol Chem. 2016;291:2974–87.

    CAS  PubMed  Article  Google Scholar 

  30. Luoh SM, Stefanich E, Solar G, Steinmetz H, Lipari T, Pestina TI, et al. Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo. Mol Cell Biol. 2000;20:507–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Garbati MR, Welgan CA, Landefeld SH, Newell LF, Agarwal A, Dunlap JB, et al. Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism. Am J Hematol. 2016;91:211–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Arshad N, Cresswell P. Tumor-associated calreticulin variants functionally compromise the peptide loading complex and impair its recruitment of MHC-I. J Biol Chem. 2018;293:9555–69.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Pecquet C, Balligand T, Chachoua I, Roy A, Vertenoeil G, Colau D, et al. Secreted mutant calreticulins as rogue cytokines trigger thrombopoietin receptor activation specifically in CALR mutated cells: perspectives for mpn therapy. Blood. 2018;132:4.

    Article  Google Scholar 

  34. Pecquet C, Chachoua I, Roy A, Balligand T, Vertenoeil G, Defour J-P, et al. MPN Calr mutants promote cell-surface localization of tpor which is obligatory for oncogenesis: novel therapeutic avenues and rescue of congenital thrombocytopenia TpoR mutants. EHA Learning Center. 2018:215926. https://learningcenter.ehaweb.org/eha/2018/stockholm/215926/christian.pecquet.mpn.calr.mutants.promote.cell-surface.localization.of.tpor.html?f=topic=1602*media=3*search=pecquet*listing=6*browseby=8.

  35. Mesaeli N, Nakamura K, Zvaritch E, Dickie P, Dziak E, Krause KH, et al. Calreticulin is essential for cardiac development. J Cell Biol. 1999;144:857–68.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Rauch F, Prud’homme J, Arabian A, Dedhar S, St-Arnaud R. Heart, brain, and body wall defects in mice lacking calreticulin. Exp Cell Res. 2000;256:105–11.

    CAS  PubMed  Article  Google Scholar 

  37. Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD, et al. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood. 2014;123:1544–51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH, et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia. 2014;28:1472–7.

    CAS  PubMed  Article  Google Scholar 

  39. Michalak M, Corbett EF, Mesaeli N, Nakamura K, Opas M. Calreticulin: one protein, one gene, many functions. Biochem J. 1999;344:281–92.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Perry JS, Hsieh CS. Development of T-cell tolerance utilizes both cell-autonomous and cooperative presentation of self-antigen. Immunol Rev. 2016;271:141–55.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Hartley SB, Cooke MP, Fulcher DA, Harris AW, Cory S, Basten A, et al. Elimination of self-reactive B lymphocytes proceeds in two stages: Arrested development and cell death. Cell. 1993;72:325–35.

    CAS  PubMed  Article  Google Scholar 

  42. Tubb VM, Schrikkema DS, Croft NP, Purcell AW, Linnemann C, Freriks MR, et al. Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies. J Immunother Cancer. 2018;6:70.

    PubMed  PubMed Central  Article  Google Scholar 

  43. Guo L, Nakamura K, Lynch J, Opas M, Olson EN, Agellon LB, et al. Cardiac-specific expression of calcineurin reverses embryonic lethality in calreticulin-deficient mouse. J Biol Chem. 2002;277:50776–9.

    CAS  PubMed  Article  Google Scholar 

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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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Balligand, T., Achouri, Y., Pecquet, C. et al. 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. Leukemia 34, 510–521 (2020). https://doi.org/10.1038/s41375-019-0538-1

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