Mutant calreticulin (CALR) resulting from a frameshift mutation becomes a “rogue cytokine,” pathologically activating the thrombopoietin receptor (TpoR), driving proliferation of hematopoietic cells in patients with myeloproliferative neoplasms (MPNs), according to research published in Blood. The findings suggest the level of circulating mutant CALR proteins could potentially be used as a biomarker for risk stratification of CALR-mutated MPNs.

The researchers used a combination of molecular and cell-based assays as well as microscopy studies to evaluate whether secreted mutant CALRs act in a paracrine or autocrine manner to activate the TpoR of adjacent cells and whether this “rogue cytokine” effect is relevant to MPN pathogenesis.

Using 159 plasma samples from patients with MPN, the researchers discovered that mutant CALR proteins are secreted. They detected soluble mutant CALR in 106 of 111 (95%) patients at levels up to 160 ng/mL in patient plasma, with a mean level of 25.64 ng/mL.


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In cases where mutational CALR burden and clinical diagnosis data were available (n=57), the team demonstrated that plasma levels of soluble mutant CALR were directly correlated with the allele burdens in the patients’ blood (r2=0.43; P <.0001).

To study the functional relevance of extracellular mutant CALR, the researchers immunoprecipitated the plasma from 5 patients with MPN and 5 control participants without MPN and analyzed the precipitating proteins using mass spectrometry. They found that plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1), and that this carrier protein complex increases mutant CALR half-life.

They went on to demonstrate that recombinant mutant CALR proteins are able to bind and activate the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation.

Using an assay able to assess protein interactions in cultured cells (HEK293), the researchers demonstrated that mutant CALR proteins produced in 1 cell specifically interact with the TpoR on different target cell. Compared with cells that only carry TpoR, they found that cells carrying both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR and respond to levels of mutant CALR that are similar to those found in patient plasma.

To test the cytokine effect of CALR, the team treated primary cells from patients with MPN carrying CALR mutations or JAK2 V617F or from healthy control participants with mutant or wildtype CALR. They demonstrated that the secreted mutant CALR induces a significant increase in megakaryocyte colony formation for patients with CALR mutations but not for patients with JAK2 V617F or healthy control participants.

“To our knowledge, this work is the first to demonstrate that circulating mutant CALR proteins can exert a rogue cytokine activity on cells that express the TpoR,” the authors wrote in their report. “Our finding that mutant CALR proteins act as rogue cytokines could open new perspectives for treating patients with CALR-mutated MPNs.”

Disclosure: Some study authors declared affiliations with biotech, pharmaceutical, or device companies. Please see the original reference for a full list of authors’ disclosures. 

Reference

Pecquet C, Papadopoulos N, Balligand T, et al. Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms. Blood. 2023;141(8):917-929. doi:10.1182/blood.2022016846