[en] Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino-acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160ng/mL, with a mean of 25.64ng/mL. Plasma mutant CALR is found in complex with soluble Transferrin Receptor 1 (sTFRC) that acts as a carrier protein and increases CALR mutant half-life. Recombinant mutant CALR proteins bound and activated the TpoR on cell lines and primary megakaryocytic progenitors from CALR-mutated patients where they drive Tpo-independent colony formation. Importantly, the CALR-sTFRC complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in one cell can specifically interact in trans with the TpoR on a target cell. Cells that carry both TpoR and mutant CALR are hypersensitive to exogenous CALR mutant proteins in comparison to cells that only carry TpoR, and respond to levels of CALR mutant proteins similar to those in patient plasma. This is consistent with CALR mutated cells exposing at the cell-surface TpoR carrying immature N-linked sugars. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.
Disciplines :
Hematology
Author, co-author :
Pecquet, Christian ; de Duve Institute, Université catholique de Louvain, Ludwig Cancer Research Brussels, Brussels, Belgium
Papadopoulos, Nicolas ; Ludwig Institute for Cancer Research, Brussels, Belgium
Balligand, Thomas ; Ludwig Institute for Cancer Research, and Université Catholique de Louvain, de Duve Institute, Woluwe-Saint-Lambert, Belgium
Chachoua, Ilyas ; Ludwig Institute for Cancer Research, Belgium
Tisserand, Amandine; INSERM, Unité Mixte de Recherche 1287, Institut Gustave Roussy, Villejuif, France
Vertenoeil, Gaëlle ; Centre Hospitalier Universitaire de Liège - CHU > > Service d'hématologie clinique ; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium
Nédélec, Audrey; Université catholique de Louvain and de Duve Institute, Belgium
Vertommen, Didier ; de Duve Institute, Brussels, Belgium
Roy, Anita ; Indian Institute of Technology Delhi, New Delhi, India
Marty, Caroline ; INSERM UMR1287, Gustave Roussy, Villejuif, France
Nivarthi, Harini; CeMM Research Centre for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Vienna, Austria
Defour, Jean-Philippe; Ludwig Institute for Cancer Research, Bruxelles, Belgium
El-Khoury, Mira; Gustave Roussy, Villejuif, France
Hug, Eva; Myelopro Diagnostics and Research GmbH, Vienna, Austria
Majoros, Andrea; MyeloPro Diagnostics and Research, Austria
Xu, Erica; MyeloPro Diagnostics and Research GmbH, Vienna, Austria
Zagrijtschuk, Oleh; Dr. med. Oleh Zagrijtschuk, MSc, Vienna, Austria
Fertig, Tudor Emanuel ; National Institute of Pathology Victor Babeș, Bucharest, Romania
Marta, Daciana S ; National Institute of Pathology Victor Babeș, Bucharest, Romania
Gisslinger, Heinz; Medical University of Vienna, Vienna, Austria
Gisslinger, Bettina; Medical University of Vienna, Vienna, Austria
Schalling, Martin; St Anna Children's Hospital, Vienna, Austria
Casetti, Ilaria Carola ; University of Pavia, Pavia, Italy
Rumi, Elisa ; University of Pavia, Pavia, Italy
Pietra, Daniela ; Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
Cavalloni, Chiara; Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
Arcaini, Luca; Division of Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
Cazzola, Mario ; University of Pavia, Pavia, Italy
Komatsu, Norio ; Juntendo University School of Medicine, Tokyo, Japan
Kihara, Yoshihiko; Juntendo University Graduate School of Medicine, Tokyo, Japan
Sunami, Yoshitaka; Juntendo university school of medicine, Bunkyo-ku, Japan
Edahiro, Yoko; Juntendo University School of Medicine, Tokyo, Japan
Araki, Marito ; Juntendo University School of Medicine, Tokyo, Japan
Lesyk, Roman ; Medical College, University of Information Technology and Management in Rzeszow, Sucharskiego, Poland
Buxhofer-Ausch, Veronika ; Ordensklinikum Linz Elisabethinen, Austria
Heibl, Sonja ; Klinikum Wels-Grieskirchen, Wels, Austria
Pasquier, Florence; Gustave Roussy, Villejuif, France
Plo, Isabelle ; INSERM UMR 1287, Villejuif, France
Vainchenker, William ; Gustave Roussy, Villejuif, France
Kralovics, Robert; Medical University of Vienna, Austria
Constantinescu, Stefan N ; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium., Brussels, Belgium
Nangalia, J, Massie, CE, Baxter, EJ, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 369:25 (2013), 2391–2405.
Klampfl, T, Gisslinger, H, Harutyunyan, AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 369:25 (2013), 2379–2390.
Michalak, M, Groenendyk, J, Szabo, E, Gold, LI, Opas, M, Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem J 417:3 (2009), 651–666.
Chachoua, I, Pecquet, C, El-Khoury, M, et al. Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. Blood 127:10 (2016), 1325–1335.
Nivarthi, H, Chen, D, Cleary, C, et al. Thrombopoietin receptor is required for the oncogenic function of CALR mutants. Leukemia 30:8 (2016), 1759–1763.
Elf, S, Abdelfattah, NS, Baral, AJ, et al. Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN. Blood 131:7 (2018), 782–786.
Elf, S, Abdelfattah, NS, Chen, E, et al. Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation. Cancer Discov 6:4 (2016), 368–381.
Marty, C, Pecquet, C, Nivarthi, H, et al. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. Blood 127:10 (2016), 1317–1324.
Araki, M, Yang, Y, Masubuchi, N, et al. Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms. Blood 127:10 (2016), 1307–1316.
Balligand, T, Achouri, Y, Pecquet, C, et al. Pathologic activation of thrombopoietin receptor and JAK2-STAT5 pathway by frameshift mutants of mouse calreticulin. Leukemia 30:8 (2016), 1775–1778.
Han, L, Schubert, C, Kohler, J, et al. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion. J Hematol Oncol, 9(1), 2016, 45.
Pecquet, C, Chachoua, I, Roy, A, et al. Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants. Blood 133:25 (2019), 2669–2681.
Araki, M, Yang, Y, Imai, M, et al. Homomultimerization of mutant calreticulin is a prerequisite for MPL binding and activation. Leukemia 33:1 (2018), 122–131.
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 34:2 (2020), 510–521.
Pecquet, C, Balligand, T, Chachoua, I, et al. Secreted mutant calreticulins as rogue cytokines trigger thrombopoietin receptor activation specifically in CALR mutated cells: perspectives for MPN therapy. Blood, 132(suppl 1), 2018, 4.
Liu, P, Zhao, L, Loos, F, et al. Immunosuppression by mutated calreticulin released from malignant cells. Mol Cell 77:4 (2019), 748–760.
Benlabiod, C, Cacemiro, MdC, Nédélec, A, et al. Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN. Nat Commun, 11(1), 2020, 4886.
Dupuis, M, Schaerer, E, Krause, KH, Tschopp, J, The calcium-binding protein calreticulin is a major constituent of lytic granules in cytolytic T lymphocytes. J Exp Med 177:1 (1993), 1–7.
Eggleton, P, Lieu, TS, Zappi, EG, et al. Calreticulin is released from activated neutrophils and binds to C1q and mannan-binding protein. Clin Immunol Immunopathol 72:3 (1994), 405–409.
Ogden, CA, deCathelineau, A, Hoffmann, PR, et al. C1q and mannose binding lectin engagement of cell surface calreticulin and Cd91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 194:6 (2001), 781–796.
Gardai, SJ, McPhillips, KA, Frasch, SC, et al. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123:2 (2005), 321–334.
Feng, M, Chen, JY, Weissman-Tsukamoto, R, et al. Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk. Proc Natl Acad Sci U S A 112:7 (2015), 2145–2150.
Beguin, Y, Soluble transferrin receptor for the evaluation of erythropoiesis and iron status. Clin Chim Acta 329:1 (2003), 9–22.
Hayes, GR, Williams, A, Costello, CE, Enns, CA, Lucas, JJ, The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide. Glycobiology 5:2 (1995), 227–232.
Rowland, M, Tozer, TN, Exposure and response after a single dose. Clinical Pharmacokinetics/Pharmacodynamics, 2005, Lippincott Williams and Wilkins, Philadelphia.
Thomas, GR, Thibodeaux, H, Errett, CJ, et al. In vivo biological effects of various forms of thrombopoietin in a murine model of transient pancytopenia. Stem Cells 14:suppl 1 (1996), 246–255.
Zhao, X, Feng, X, Wu, Z, et al. Persistent elevation of plasma thrombopoietin levels after treatment in severe aplastic anemia. Exp Hematol 58 (2018), 39–43.
Wood, TJ, Sliva, D, Lobie, PE, et al. Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter. Mol Cell Endocrinol 130:1-2 (1997), 69–81.
Wang, S, He, X, Wu, Q, et al. Transferrin receptor 1-mediated iron uptake plays an essential role in hematopoiesis. Haematologica 105:8 (2020), 2071–2082.
Drachman, JG, Millett, KM, Kaushansky, K, Thrombopoietin signal transduction requires functional JAK2, not TYK2. J Biol Chem 274:19 (1999), 13480–13484.
Michalak, M, Corbett, EF, Mesaeli, N, Nakamura, K, Opas, M, Calreticulin: one protein, one gene, many functions. Biochem J 344:2 (1999), 281–292.
Kapoor, M, Ellgaard, L, Gopalakrishnapai, J, et al. Mutational analysis provides molecular insight into the carbohydrate-binding region of calreticulin: pivotal roles of tyrosine-109 and aspartate-135 in carbohydrate recognition. Biochemistry 43:1 (2004), 97–106.
Greenbaum, HS, Evers, M, Rosencrance, A, et al. Type I calreticulin mutations result in hyperactivation of its acetyltransferase function and iron metabolism, inducing a susceptibility to ferroptosis. Blood, 138(suppl 1), 2021, 3593.
Achyutuni, S, Nivarthi, H, Majoros, A, et al. Hematopoietic expression of a chimeric murine-human CALR oncoprotein allows the assessment of anti-CALR antibody immunotherapies in vivo. Am J Hematol 96:6 (2021), 698–707.
