Secreted Mutant Calreticulins As Rogue Cytokines in Myeloproliferative Neoplasms.

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.