Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.
Subscribe to Journal
Get full journal access for 1 year
only $9.98 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
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.
Cabagnols X, Favale F, Pasquier F, Messaoudi K, Defour JP, Ianotto JC, et al. Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients. Blood. 2016;127:333–42.
Milosevic Feenstra JD, Nivarthi H, Gisslinger H, Leroy E, Rumi E, Chachoua I, et al. Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms. Blood. 2016;127:325–32.
Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006;3:e270.
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.
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.
Cabagnols X, Defour JP, Ugo V, Ianotto JC, Mossuz P, Mondet J, et al. Differential association of calreticulin type 1 and type 2 mutations with myelofibrosis and essential thrombocytemia: relevance for disease evolution. Leukemia. 2015;29:249–52.
Tefferi A, Wassie EA, Guglielmelli P, Gangat N, Belachew AA, Lasho TL, et al. Type 1 versus Type 2 calreticulin mutations in essential thrombocythemia: a collaborative study of 1027 patients. Am J Hematol. 2014;89:E121–4.
Eder-Azanza L, Navarro D, Aranaz P, Novo FJ, Cross NC, Vizmanos JL. Bioinformatic analyses of CALR mutations in myeloproliferative neoplasms support a role in signaling. Leukemia. 2014;28:2106–9.
Pietra D, Rumi E, Ferretti VV, Di Buduo CA, Milanesi C, Cavalloni C, et al. Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms. Leukemia. 2016;30:431–8.
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.
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.
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.
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.
Balligand T, Achouri Y, Pecquet P, Gaudray G, Colau D, Hug E, et al. Knock-in of Murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr Exon9 in Cardiac Development. Leukemia. 2020;34:510–21.
Li J, Prins D, Park HJ, Grinfeld J, Gonzalez-Arias C, Loughran S, et al. Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage. Blood. 2018;131:649–61.
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. 2017;31:1136–44.
Andrikovics H, Krahling T, Balassa K, Halm G, Bors A, Koszarska M, et al. Distinct clinical characteristics of myeloproliferative neoplasms with calreticulin mutations. Haematologica. 2014;99:1184–90.
Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L, et al. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood. 2014;123:1552–5.
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.
Tefferi A, Lasho TL, Finke C, Belachew AA, Wassie EA, Ketterling RP, et al. Type 1 vs type 2 calreticulin mutations in primary myelofibrosis: differences in phenotype and prognostic impact. Leukemia. 2014;28:1568–70.
Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127:2391–405.
Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia. 2014;28:1407–13.
Allen C, Lambert JR, Linch DC, Gale RE. X chromosome inactivation analysis reveals a difference in the biology of ET patients with JAK2 and CALR mutations. Blood. 2014;124:2091–3.
Dupont S, Masse A, James C, Teyssandier I, Lecluse Y, Larbret F, et al. The JAK2 617V>F mutation triggers erythropoietin hypersensitivity and terminal erythroid amplification in primary cells from patients with polycythemia vera. Blood. 2007;110:1013–21.
Scott LM, Scott MA, Campbell PJ, Green AR. Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera, but not essential thrombocythemia. Blood. 2006;108:2435–7.
Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA. New mutations and pathogenesis of myeloproliferative neoplasms. Blood. 2011;118:1723–35.
Kjaer L, Holmstrom MO, Cordua S, Andersen MH, Svane IM, Thomassen M, et al. Sorted peripheral blood cells identify CALR mutations in B- and T-lymphocytes. Leuk Lymphoma. 2018;59:973–7.
Delhommeau F, Dupont S, Tonetti C, Masse A, Godin I, Le Couedic JP, et al. Evidence that the JAK2 G1849T (V617F) mutation occurs in a lymphomyeloid progenitor in polycythemia vera and idiopathic myelofibrosis. Blood. 2007;109:71–7.
Nam AS, Kim KT, Chaligne R, Izzo F, Ang C, Taylor J, et al. Somatic mutations and cell identity linked by Genotyping of Transcriptomes. Nature. 2019;571:355–60.
Rodriguez-Meira A, Buck G, Clark SA, Povinelli BJ, Alcolea V, Louka E, et al. Unravelling intratumoral heterogeneity through high-sensitivity single-cell mutational analysis and parallel RNA sequencing. Mol Cell. 2019;73:1292–305. e8.
Anand S, Stedham F, Beer P, Gudgin E, Ortmann CA, Bench A, et al. Effects of the JAK2 mutation on the hematopoietic stem and progenitor compartment in human myeloproliferative neoplasms. Blood. 2011;118:177–81.
James C, Mazurier F, Dupont S, Chaligne R, Lamrissi-Garcia I, Tulliez M, et al. The hematopoietic stem cell compartment of JAK2V617F-positive myeloproliferative disorders is a reflection of disease heterogeneity. Blood. 2008;112:2429–38.
Kent DG, Li J, Tanna H, Fink J, Kirschner K, Pask DC, et al. Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion. PLoS Biol. 2013;11:e1001576.
Saint-Martin C, Leroy G, Delhommeau F, Panelatti G, Dupont S, James C, et al. Analysis of the ten-eleven translocation 2 (TET2) gene in familial myeloproliferative neoplasms. Blood. 2009;114:1628–32.
Ortmann CA, Kent DG, Nangalia J, Silber Y, Wedge DC, Grinfeld J, et al. Effect of mutation order on myeloproliferative neoplasms. N Engl J Med. 2015;372:601–12.
Delhommeau F, Dupont S, Della Valle V, James C, Trannoy S, Masse A, et al. Mutation in TET2 in myeloid cancers. N. Engl J Med. 2009;360:2289–301.
Lundberg P, Karow A, Nienhold R, Looser R, Hao-Shen H, Nissen I, et al. Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms. Blood. 2014;123:2220–8.
Passamonti F, Rumi E, Pietra D, Elena C, Boveri E, Arcaini L, et al. A prospective study of 338 patients with polycythemia vera: the impact of JAK2 (V617F) allele burden and leukocytosis on fibrotic or leukemic disease transformation and vascular complications. Leukemia. 2010;24:1574–9.
Rumi E, Pietra D, Guglielmelli P, Bordoni R, Casetti I, Milanesi C, et al. Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms. Blood. 2013;121:4388–95.
Barosi G, Rosti V, Bonetti E, Campanelli R, Carolei A, Catarsi P, et al. Evidence that prefibrotic myelofibrosis is aligned along a clinical and biological continuum featuring primary myelofibrosis. PLoS ONE. 2012;7:e35631.
Campbell PJ, Scott LM, Buck G, Wheatley K, East CL, Marsden JT, et al. Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet. 2005;366:1945–53.
Kollmann K, Warsch W, Gonzalez-Arias C, Nice FL, Avezov E, Milburn J, et al. A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation. Leukemia. 2017;31:934–44.
Chagraoui H, Komura E, Tulliez M, Giraudier S, Vainchenker W, Wendling F. Prominent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice. Blood. 2002;100:3495–503.
Lau WW, Hannah R, Green AR, Gottgens B. The JAK-STAT signaling pathway is differentially activated in CALR-positive compared with JAK2V617F-positive ET patients. Blood. 2015;125:1679–81.
Di Buduo CA, Abbonante V, Marty C, Moccia F, Rumi E, Pietra D, et al. Defective interaction of mutant calreticulin and SOCE in megakaryocytes from patients with myeloproliferative neoplasms. Blood. 2020;135:133–44.
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.
Pronier E, Cifani P, Merlinsky TR, Berman KB, Somasundara AVH, Rampal RK, et al. Targeting the CALR interactome in myeloproliferative neoplasms. JCI Insight. 2018;3:e122703.
Mondet J, Park JH, Menard A, Marzac C, Carillo S, Pourcelot E, et al. Endogenous megakaryocytic colonies underline association between megakaryocytes and calreticulin mutations in essential thrombocythemia. Haematologica. 2015;100:e176–8.
Gekas C, Graf T. CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age. Blood. 2013;121:4463–72.
Sanjuan-Pla A, Macaulay IC, Jensen CT, Woll PS, Luis TC, Mead A, et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature. 2013;502:232–6.
Kjaer L, Cordua S, Holmstrom MO, Thomassen M, Kruse TA, Pallisgaard N, et al. Differential dynamics of CALR mutant Allele burden in myeloproliferative neoplasms during Interferon Alfa Treatment. PLoS ONE. 2016;11:e0165336.
Debili N, Coulombel L, Croisille L, Katz A, Guichard J, Breton-Gorius J, et al. Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow. Blood. 1996;88:1284–96.
This work was funded by grants from the Agence Nationale pour la Recherche (Thrombocytosis, WV), the Ligue Nationale contre le Cancer (équipe labellelisée HR 2016, 2019), the Fondation Laurette Fugain (WV, IP), INCA PLBIO 2016 (IP), by grant “ Taxe d’apprentissage” Gustave Roussy—2016 (LS and MEK) and from the INSERM. Labex GR-Ex (IP, WV) is funded by the program “Investissements d’avenir.” Support to SNC was from Fondation contre le cancer, Salus Sanguinis Action de recherché concertée (ARC) 16/21-073 and WelBio F 44/8/5—MCF/UIG—10955. MEK was supported by the SFH (Société française d’hématologie), XC and AT were supported by the university Paris-Diderot (MENRT grant), MM by la Ligue Nationale contre le Cancer, Gustave Roussy Foundation and SFH. GRM was a recipient from allocations doctorales sur domaines cibles (ARDoc, Ile-de-France region). GV was supported by a PhD aspirant fellowship of the Fonds Nationals de la Recherche Scientifique, Belgium. We want to thank the platforms of bioinformatic (Khadija M Diop, Guillaume Meurice and Marc Deloger) and flow cytometry and Edwige Leclercq, Marie-Hélène Courtier for CALR sizing analysis.
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
El-Khoury, M., Cabagnols, X., Mosca, M. et al. Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms. Oncogene 39, 5323–5337 (2020). https://doi.org/10.1038/s41388-020-1368-3