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Updated: Aug 22, 2025
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms
Christian Pecquet1,2, Nicolas Papadopoulos1,2, Thomas Balligand1,2
1Ludwig Cancer Research, Brussels, Belgium.
Abstract:
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 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. 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.
Insights
Mutant calreticulin (CALR) proteins secreted from cancer cells activate the thrombopoietin receptor (TpoR), driving myeloproliferative neoplasms (MPNs). These mutant CALR proteins, carried by sTFR1, specifically target MPN cells, acting as rogue cytokines.
Area of Science:
- Molecular Biology
- Oncology
- Hematology
Background:
- Somatic mutations in the CALR gene, specifically a -1/+2 frameshift in exon 9, produce mutant calreticulin (CALR) proteins.
- These mutant CALR proteins possess a novel C-terminal amino acid sequence and are implicated in the pathogenesis of myeloproliferative neoplasms (MPNs).
- Previous studies indicated that mutant CALRs interact with and activate the thrombopoietin receptor (TpoR/MPL) within the same cell.
Purpose of the Study:
- To investigate the secretion of mutant CALR proteins and their functional consequences on TpoR activation.
- To determine the role of soluble transferrin receptor 1 (sTFR1) in the stability and function of circulating mutant CALR.
- To elucidate the mechanism by which secreted mutant CALR proteins specifically target MPN-driving clones.
Main Methods:
- Quantification of mutant CALR proteins in patient plasma.
- Biochemical assays to assess the interaction of mutant CALR with sTFR1 and TpoR.
- Functional studies using cell lines and primary megakaryocytic progenitors to evaluate TpoR activation and cell proliferation.
- Bioluminescence resonance energy transfer (BRET) assays to study intercellular interactions.
Main Results:
- Mutant CALR proteins are secreted into patient plasma at significant levels (mean 25.64 ng/mL).
- Plasma mutant CALR forms a complex with sTFR1, which enhances its half-life and maintains TpoR activating function.
- Secreted mutant CALR proteins activate TpoR in a cell-autonomous and cell-non-autonomous manner, leading to thrombopoietin-independent megakaryopoiesis.
- MPN cells expressing TpoR exhibit hypersensitivity to exogenous mutant CALR, particularly when TpoR carries immature N-linked sugars.
Conclusions:
- Mutant CALR proteins function as secreted "rogue cytokines" that drive MPN development.
- The CALR-sTFR1 complex is a key circulating factor in MPN pathogenesis.
- Secreted mutant CALR proteins specifically target and activate TpoR on MPN clone cells, highlighting a potential therapeutic vulnerability.
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