Characterization of secretion signals within the novel carboxy-terminal domain of mutant calreticulin in myeloproliferative neoplasms

Abstract

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders marked by excessive production of mature myeloid cells. In a subset of patients, mutations in calreticulin (CALR) drive the disease by constitutive activation of the JAK/STAT pathway. All CALR mutations result in a +1 frameshift that generates a novel carboxy- terminal sequence enriched in basic residues and lacking the KDEL ER-retention signal. While CALR normally resides in the endoplasmic reticulum as a multifunctional chaperone, in MPNs mutant CALR is aberrantly secreted, however the precise mechanism underlying this process remains not fully understood. In this study, we aimed to investigate the secretion mechanism of CALR mutant to better understand its role in MPN development. To this end, we employed both in silico and wet lab analysis such as ELM and CELLO prediction tools, site directed mutagenesis and Western blotting techniques. Contrary to the current understanding suggesting that CALR mutant secretion is mainly caused by the loss of the KDEL retention signal, our results indicate that the secretion is largely driven by the newly generated motifs rich in arginine residues. Furthermore, prediction scores suggest that these motifs may also direct the mutant protein to other subcellular compartments, particularly the nucleus. Understanding this mechanism is crucial, as it highlights specific motifs as potential therapeutic targets that could be exploited to develop treatments for CALR-mutant MPN patients.