Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms

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 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.

The therapeutic mavericks: Potent immunomodulating chaperones capable of treating human diseases

“…Recent work has revealed cells presenting with both the TPO receptor and mutant CRT complex are hypersensitive to additional exposure to exogenously released mutant CRT. Indeed, the exogenous mutant CRT found at levels observed in MPN patients, acts as a ‘rogue’ cytokine, capable of activating TPO receptor/JAK–STAT signalling in patient primary cells, by binding to the immature N‐glycosylated TPO receptor complexed with mutant endogenous mutant CRT 61 …”

“…Indeed, the exogenous mutant CRT found at levels observed in MPN patients, acts as a ‘rogue’ cytokine, capable of activating TPO receptor/JAK–STAT signalling in patient primary cells, by binding to the immature N‐glycosylated TPO receptor complexed with mutant endogenous mutant CRT. 61 …”

Effects of calreticulin mutations on cell transformation and immunity

“…This results in mutant CRT localization on the cell surface 14 , 15 , 16 and the extracellular space via Golgi‐mediated secretion. 15 , 17 , 18 , 19 …”

“… 15 , 19 , 52 A recent study shows that the secreted forms of CRT mutants are stabilized by binding to the soluble transferrin receptor 1 (sTFRC). 15 Paracrine activation of MPL signalling by secreted forms of CRT mutants was not observed when healthy cells expressing MPL and WT CRT were treated with supernatants from CRT Del52 expressing cells. 18 However, a recent study demonstrates that exogenous recombinant CRT Del52 promotes cytokine‐independent proliferation, particularly of cells expressing both mutant CRT and MPL.…”

“… 18 However, a recent study demonstrates that exogenous recombinant CRT Del52 promotes cytokine‐independent proliferation, particularly of cells expressing both mutant CRT and MPL. 15 Cells expressing both MPL and mutant CRTs expose immature MPL glycans on the cell surface to induce additional activation by exogenously supplied CRT mutants. 15 …”