In this issue of Blood, Marty et al, Chachoua et al, and Araki et al report results of studies unraveling the molecular pathogenesis of CALR-mutant myeloproliferative neoplasms (MPNs). Together, these 3 reports define a novel disease paradigm, whereby a mutant chaperone constitutively activates receptor signaling through an abnormal interaction with the thrombopoietin (TPO) receptor (MPL).1-3 

Schematic representation of the molecular pathogenesis of CALR-mutant MPNs based on the findings of the 3 articles of this issue.1-3  (A) TPO-independent activation of the TPO receptor (MPL) by mutant calreticulin in an individual hematopoietic cell. (i) Abnormal physical interaction between mutant calreticulin and TPO receptor within the ER. (ii) MPL coupled with mutant calreticulin is exported on the cell surface through the Golgi apparatus and/or an alternative pathway. (iii) TPO-independent activation of MPL, and in turn dimerization and activation of JAK2. As a chaperone, calreticulin normally assists proper folding of the TPO receptor (MPL) inside the ER. Mutant calreticulin preferentially interacts with MPL, and the mutant-specific domain of the protein is required for this interaction that leads to stable binding. Here, it is assumed that the MPL dimer is already assembled in the ER, but another possibility is that dimerization occurs on the cell surface. It is also uncertain whether both MPL subunits bind mutant calreticulin, or just one of them does; again, both events might take place. The interaction of mutant calreticulin with MPL activates MPL signaling through JAK2. (B) Biological and clinical consequences. (i) Activated MPL-driven clonal expansion of HSCs. (ii) Excessive platelet production by abnormal megakaryocytes: ET. (iii) Bone marrow reticulin formation: progression from ET to myelofibrosis. Most patients with a CALR-mutant MPN have fully clonal hematopoiesis, indicating that the founding mutation drives clonal expansion at the HSC level. Under normal conditions, adult HSCs are TPO-dependent for their survival and maintenance in the osteoblastic niche10,11 ; thus, mutant calreticulin-induced abnormal MPL signaling is likely responsible for clonal expansion of HSCs carrying a CALR mutation. At the hematopoietic precursor level, the only cells in which MPL signaling is effective are megakaryocytes, and therefore MPL-signaling activation leads to overproduction of platelets. Mutant calreticulin-expressing megakaryocytes also contribute to bone marrow fibrosis, and in the long-term this process leads to a transition from ET to myelofibrosis. Professional illustration by Patrick Lane, ScEYEnce Studios.

Schematic representation of the molecular pathogenesis of CALR-mutant MPNs based on the findings of the 3 articles of this issue.1-3  (A) TPO-independent activation of the TPO receptor (MPL) by mutant calreticulin in an individual hematopoietic cell. (i) Abnormal physical interaction between mutant calreticulin and TPO receptor within the ER. (ii) MPL coupled with mutant calreticulin is exported on the cell surface through the Golgi apparatus and/or an alternative pathway. (iii) TPO-independent activation of MPL, and in turn dimerization and activation of JAK2. As a chaperone, calreticulin normally assists proper folding of the TPO receptor (MPL) inside the ER. Mutant calreticulin preferentially interacts with MPL, and the mutant-specific domain of the protein is required for this interaction that leads to stable binding. Here, it is assumed that the MPL dimer is already assembled in the ER, but another possibility is that dimerization occurs on the cell surface. It is also uncertain whether both MPL subunits bind mutant calreticulin, or just one of them does; again, both events might take place. The interaction of mutant calreticulin with MPL activates MPL signaling through JAK2. (B) Biological and clinical consequences. (i) Activated MPL-driven clonal expansion of HSCs. (ii) Excessive platelet production by abnormal megakaryocytes: ET. (iii) Bone marrow reticulin formation: progression from ET to myelofibrosis. Most patients with a CALR-mutant MPN have fully clonal hematopoiesis, indicating that the founding mutation drives clonal expansion at the HSC level. Under normal conditions, adult HSCs are TPO-dependent for their survival and maintenance in the osteoblastic niche10,11 ; thus, mutant calreticulin-induced abnormal MPL signaling is likely responsible for clonal expansion of HSCs carrying a CALR mutation. At the hematopoietic precursor level, the only cells in which MPL signaling is effective are megakaryocytes, and therefore MPL-signaling activation leads to overproduction of platelets. Mutant calreticulin-expressing megakaryocytes also contribute to bone marrow fibrosis, and in the long-term this process leads to a transition from ET to myelofibrosis. Professional illustration by Patrick Lane, ScEYEnce Studios.

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When Klampfl et al detected somatic mutations of calreticulin (encoded by the CALR gene) in patients with primary myelofibrosis (PMF) with wild-type JAK2 and MPL, it was difficult to understand why mutation in a housekeeping gene, required for the maintenance of basic cellular function, would cause an MPN.4  In a follow-up study of several types of myeloid neoplasms, we made the illuminating observation that somatic CALR mutations occurred almost exclusively in patients with thrombocytosis: essential thrombocythemia (ET), PMF, and the rare myelodysplastic/MPN defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis.4  This clearly established a strong causative relationship between CALR mutation and excessive platelet production, although the underlying molecular mechanism(s) involved were unclear.

Calreticulin resides in the lumen of the endoplasmic reticulum (ER), where it functions as a molecular chaperone for many glycoproteins, assisting their regular folding.5  In addition, the C-terminal domain of calreticulin is responsible for calcium-buffering activity, which controls calcium homeostasis and, in turn, signaling processes. Somatic CALR mutations result in insertions and deletions generating a frameshift, and cluster in the last exon (exon 9) of the gene.4  More than 50 different types of insertions/deletions in CALR have been detected, but a 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent types, overall found in >80% of all patients with a CALR-mutant MPN.6,7  The type 2 mutation is predominantly associated with an ET phenotype, whereas the type 1 mutation is mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in ET.6 

To study the contribution of CALR mutants to the pathogenesis of MPNs, Marty et al engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing the mutants.1  CALRdel52 (type 1 mutation)-expressing mice rapidly developed marked thrombocytosis and then progressed to a condition similar to human myelofibrosis. By contrast, CALRins5 (type 2 mutation)-expressing mice had a mild ET phenotype with low propensity to disease progression. Thus, both mutants specifically amplified the megakaryocyte lineage and increased platelet production. It should be noted that Pietra et al8  have recently reported differential clinical effects of different mutation subtypes in CALR-mutant MPNs, and the clinical effects are very similar to those observed by Marty et al1  in their murine models. Using a cell line model, Marty et al then demonstrate that CALR mutants specifically activate MPL to induce constitutive activation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5/3/1 (STAT5/3/1).1  In addition, they show that CALRdel52 cannot induce thrombocytosis in Mpl knockout mice. Altogether, these observations indicate that CALR mutants are sufficient to induce thrombocytosis through MPL activation.

Using cell line–based transcriptional and proliferation assays, Chachoua et al assessed the ability of CALR mutants to induce activation of a series of cytokine receptors that signal via the JAK/STAT pathway.2  They found that pathogenic CALR mutants specifically activate the TPO receptor by a mechanism dependent on the presence of the extracellular N-glycosylation residues of MPL and the glycan-binding site at the new C-terminal tail of the mutant calreticulin. Expression of a soluble extracellular MPL domain blocks pathological signaling of CALR mutants via the TPO receptor, provided it is N-glycosylated. Signaling induced by CALR mutants via MPL directly leads to dimerization and activation of JAK2, and downstream STAT5/3/1, mitogen-activated protein kinase, and phosphatidylinositol-3 kinase signaling. Chachoua et al also studied patient cells using specific short hairpin RNAs, which allowed them to confirm the crucial role of MPL and JAK2 in CALR mutant-induced spontaneous growth of megakaryocytic progenitors.2 

Araki et al used the TPO-dependent megakaryocytic cell line UT-7/TPO previously generated by their laboratory.3  Using this cell line, they demonstrate that mutant, but not wild-type, CALR activates MPL and downstream signaling molecules including JAK2, STAT5, and extracellular signal-regulated kinase 1/2, and subsequently promotes the TPO‐independent growth of UT‐7/TPO cells. Araki et al also provide evidence that mutant calreticulin preferentially binds to MPL, and that the mutant‐specific domain of calreticulin is required for this interaction.3  Additionally, they show the cell surface localization of mutant calreticulin with no paracrine activation capacity. Finally, they demonstrate that MPL is required for TPO‐independent megakaryocytopoiesis in induced pluripotent stem cell–derived hematopoietic stem cells (HSCs) harboring a CALR mutation.

A late-breaking abstract at the 2015 American Society of Hematology meeting reported findings indicating that physical interaction between mutant calreticulin and the TPO receptor is required for hematopoietic transformation and development of MPNs.9 

The figure summarizes the currently available evidence concerning the molecular pathogenesis of CALR-mutant MPNs. The interaction between mutant calreticulin and the TPO receptor likely occurs within the ER, where chaperoning of proteins takes place.5  MPL coupled with mutant calreticulin is then exported to the cell surface, and this liaison results in constitutive activation of mutant megakaryocytes. Many patients with a CALR-mutant MPN have a granulocyte mutant allele burden of ∼40% to 50%, indicating that they have fully clonal hematopoiesis. This suggests that activation of MPL must provide an advantage at the stem cell level, consistent with the crucial role of TPO and MPL in maintaining adult quiescent HSCs.10,11  At the hematopoietic precursor level, the only cells that are activated by MPL signaling are megakaryocytes, and therefore CALR-mutant clonal hematopoiesis results in selective overproduction of platelets. Thrombocytosis is the initial phenotype of all CALR-mutant MPNs, but a portion of patients develop bone marrow fibrosis progressing to myelofibrosis with myeloid metaplasia.6  Type 1 CALR mutation involves a high risk of myelofibrotic transformation, likely because it specifically impairs the calcium-buffering activity of calreticulin in activated megakaryocytes.8 

Will our better understanding of the pathogenesis of CALR-mutant MPNs translate into better management of patients? Unfortunately, we cannot rely upon specific MPL inhibitors at present. However, a study recently published in this journal has shown that interferon α can inhibit platelet overproduction and also clonal hematopoiesis in CALR-mutant ET.12  In addition, an interesting study has recently shown that megakaryocytes drive fibrosis in animal models of MPNs, and that aurora kinase A is required for myelofibrosis development.13  This study also shows that targeting megakaryocytes with aurora kinase A inhibitors has the potential to provide therapeutic benefit. Ad hoc clinical trials are needed to evaluate whether this inhibition will be safe and effective in treating patients with myelofibrosis.

Conflict-of-interest disclosure: The author declares no competing financial interests.

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