Elucidating how mutations in a chaperone, calreticulin, induce blood cancer

11/05/2023

Since 2000, the group of Stefan Constantinescu at UCLouvain, de Duve Institute and Ludwig Institute for Cancer Research is mapping the molecular bases of a group of blood cancers, myeloproliferative neoplasms, that become more prevalent with age and that are associated with significant complications such as thromboses and evolution to severe leukemia. In recent work, the group elucidates how mutants of a protein, calreticulin (supposed to help other proteins fold) gain the ability to induce blood cancer. The latest results of the group are critical because these blood cancers are difficult to treat: these novel published findings bring these diseases closer to be amenable to treatment.

Years of research

These results are part of a long history of research conducted by Prof. Stefan Constantinescu and his team at de Duve Institute, UCLouvain and Ludwig Institute for Cancer Research Laboratories in Brussels. Since 2017, he is a Welbio investigator. 

• 2004-2005: Stefan Constantinescu hypothesized, based on cell biology data, that JAK2 pathway may be linked to human diseases myeloproliferative neoplasms, like Polycythemia Vera and Myelofibrosis. The group contributed, in collaboration with the group of William Vainchenker at Institut Gustave Roussy (Villejuif, France) to the discovery of the acquired mutation JAK2 V617F that is responsible of 70% of BCR-ABL myeloproliferative neoplasms. This work was published in Nature.
  The group also reported the first oncogenic active mutations in two other members of the Janus kinase family JAK1 and TYK2, later found in other cancers. 
   
• 2006: the group identified mutants in TpoR at position W515 which induced oncogenic activation of TpoR in the absence of its ligand, thrombopoietin. These mutants in TpoR W515 were subsequently found by others and the group to be present in a subset of MPN patients that do not harbor JAK2 V617F. The work was published in 2006 and 2010 in Blood, with structural mechanism in 2013 in Proc. Natl. Acad. Sci. USA.
   
• 2016 and 2019: Constantinescu adn his team studied another set of mutations present in 20-30% of myeloproliferative neoplasms (that do not harbor JAK2 or MPL mutations), namely mutations in the calreticulin gene (CALR). The group reported that mutant CALRs require TpoR to induce blood cancers. The work was published in 2016 in Blood and its biochemical basis in 2019 also in Blood.

Mutant calreticulins are secreted, act as rogue cytokines on cells expressing TpoR enhancing the fitness of the MPN clone.

In 2022, the group demonstrated that mutant CALR proteins are also secreted especially from cells belonging to the clone that do not also express TpoR; the circulating mutant CALR can be detected in patients, as the study centralized plasma from >100 patients from several countries. The circulating mutant CALR is secreted in complex with another protein that protects it from degradation. Strikingly, the circulating mutant CALR can bind to cells expressing TpoR and endogenous CALR mutants, stabilizing the complexes and enhancing signaling. This is a new mechanism by which a chaperone molecule, calreticulin, can be rendered oncogenic by frameshift mutations, and where the secreted protein can act as a cytokine to specifically enhance the proliferation of the MPN clone that already carries endogenous mutant CALR due to the oligomerizing abilities of the mutant CALR and immature glycosylation of TpoR only in mutated CALR cells.

The work is highly relevant for efforts to inhibit mutant CALR signaling at the surface of cells, since circulating mutant CALR may act as a sink for protein therapeutics targeting mutant CALR.

Mutant calreticulins are secreted, act as rogue cytokines on cells expressing TpoR enhancing the fitness of the MPN clone. CALR mutants are secreted in the plasma of Myeloproliferative neoplasm patients in complex with soluble TFRC (sTRFC) which stabilizes the mutated CALR. Circulating mutant CALR acts as “rogue cytokine” on neighboring mutated CALR cells expressing immature forms of TpoR leading to the activation of the TpoR signaling and subsequent amplification of the mutated clone.

The co-first authors of this study published in 2023 in Blood (a high impact journal in hematology and immunology) are Christian Pecquet, Nicolas Papadopoulos, Thomas Balligand, Ilyas Chachoua from the group and Amandine Tisserand from Institut Gustave Roussy, Villejuif, France. A major part of the work concerned patient samples from several hematology centers, and the collaboration with Prof. Violaine Havelange from Cliniques universitaires Saint Luc was extremely important.

Specificity of interaction between mutant CALRs and TpoR elucidated

In a 2023 paper just accepted for publication in Nature Communications, the group used hydrogen-deuterium exchange mass spectrometry and discovered that certain acidic patches in the extracellular domain of TpoR contact the new positively charged tail in mutant CALRs and that this interaction is key for explaining the stability and specificity of the complex formed between the two proteins. In addition, the interaction requires the immature sugars of TpoR and the N-terminal lectin domain of calreticulin, but the latter interaction occurs transiently between calreticulin and hundreds of N-glycosylated proteins. Combining biochemical and modeling approaches, a new detailed map of the interaction has been elucidated where the specificity of the interaction between mutant CALR and TpoR is provided by binding of the mutant tail to acidic patches of TpoR while the stability of this interaction is reinforced by strong interaction between immature N-glycans of TpoR and CALR mutant N-terminal lectin domain.

Specificity of interaction between mutant CALRs and TpoR elucidated CALR mutant C-terminus mediates dual binding to the Thrombopoietin Receptor (TpoR) triggering complex dimerization and activation. Frameshift mutants of CALR acquire a novel positively charged C-terminus that specifically interacts with negative patches of TpoR extracellular domain and induces dimerization of the complex. CALR is shown is grey, the mutant C-terminus is shown in blue and TpoR is shown in orange.

The first author of this work is Nicolas Papadopoulos. Using the findings published in this work, Audrey Nédélec (second author of this work) is now developing in collaboration with Nicolas Papadopoulos novel inhibitors of the CALR mutant-TpoR interaction that have the potential to become a novel treatment for patients suffering from myeloproliferative neoplasms.

The UCLouvain scientists are charting new avenues for treatment against myeloproliferative neoplasms.

Articles describing this research

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.
Pecquet C, Papadopoulos N, Balligand T, Chachoua I, Tisserand A, Vertenoeil G, Nédélec A, Vertommen D, Roy A, Marty C, Nivarthi H, Defour JP, El-Khoury M, Hug E, Majoros A, Xu E, Zagrijtschuk O, Fertig TE, Marta DS, Gisslinger H, Gisslinger B, Schalling M, Casetti I, Rumi E, Pietra D, Cavalloni C, Arcaini L, Cazzola M, Komatsu N, Kihara Y, Sunami Y, Edahiro Y, Araki M, Lesyk R, Buxhofer-Ausch V, Heibl S, Pasquier F, Havelange V, Plo I, Vainchenker W, Kralovics R, Constantinescu SN.
Blood (2023) 141(8):917-929

Introductory comment: CALR goes rogue. Melo-Cardenas J, Crispino JD. Blood (2023) 141(8):818-820

Oncogenic CALR Mutant C-terminus Mediates Dual Binding to the Thrombopoietin Receptor Triggering Complex Dimerization and Activation.
Papadopoulos N, Nédélec A, Derenne A, Şulea TA, Pecquet C, Chachoua I, Vertenoeil G, Tilmant T, Petrescu AJ, Mazzucchelli G, Iorga BI, Vertommen D, Constantinescu SN.
Nat. Comm. (2023) 14(1):1881

In the press

MediQuality.net - Vers un traitement ciblé pour un cancer du sang : le néoplasme myéloprolifératif  - 17/05/2023
DailyScience.be - Casser l’alliance qui conduit à des cancers du sang… et éviter certaines thromboses  - 22/06/2023

MediQuality.net - Naar een gerichte behandeling van een bloedkanker: myeloproliferatieve neoplasma  - 17/05/2023

Funding

Stefan Constantinescu is a Welbio investigator within the WEL Research Institute. This work was also supported by Fondation contre le Cancer, Actions de Recherche Concertées, the FNRS (Le Fonds de la Recherche Scientifique), Ludwig Institute for Cancer Research, de Duve Institute, Fondation Salus Sanguinis and Fondation Les avions de Sébastien. Nicolas Papadopoulos has received an FSR PhD Fellowship from Université catholique de Louvain and an Aspirant PhD Fellowship from the FRS-FNRS, Belgium

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