New molecular and cellular aspects of mutant calreticulin in Myeloproliferative Neoplasms

Morlan-mairal, M

Abstract

Calreticulin (CALR) is an endoplasmic reticulum (ER) protein that plays an important role as a calcium (Ca2+) buffering chaperone. Mutations in CALR exon 9 have been identified in essential thrombocythemia and primary myelofibrosis, two myeloproliferative neoplasms (MPNs) characterised by megakaryocyte hyperplasia. Despite the large body of research built around CALR mutations, many aspects of the oncogenic mechanisms of CALR in MPNs remain unanswered. This investigation aims to investigate whether CALR mutations affect the nature of the C-terminal domain of this protein, its sub-cellular compartmentalisation and its Ca2+ buffering activity during megakaryocyte hyperplasia. Additionally, this study establishes a new cellular model to investigate megakaryocyte differentiation in presence of CALR mutations.
In silico analysis of the structural characteristics of CALR mutant C-terminal domain revealed that CALR mutations lead to changes in its secondary structure, its protein binding properties and changes the acidity of CALR mutant´s C-terminal domain. These physical alterations could affect CALR cellular behaviour by leading to inefficient ER Ca2+ buffering activity and lead to a novel oncogenic network of protein interactions.
This study revealed that MARIMO leukemic cell line, which harbours a CALR mutation, has in vitro megakaryocyte differentiation potential. Importantly, this discovery was useful for further studies aiming to analyse CALR mutant cells during megakaryocyte commitment. Moreover, study of CALR mutant cellular localisation showed that this protein is localised within the ER, but it is also mislocalised within the cytoplasm and cell membrane, where it co-localised with thrombopoietin receptor. Interestingly, CALR cell surface expression increased during megakaryocyte commitment in CALR mutant cells, showing a dynamic process of CALR compartmentalisation during megakaryocyte differentiation.
One of the more significant findings shown in this study is the emergence of intracellular Ca2+ concentrations ([Ca2+I]) as an important element during megakaryopoiesis. Importantly, CALR mutations impaired the cellular ER Ca2+ buffering activity and led to changes in the [Ca2+I] during the process of megakaryocyte differentiation. In addition, initial experimentsrevealed that physical manipulation of [Ca2+I] leads to the emergence of a megakaryocyte phenotype in leukemic cells, showing the relevance of this factor during megakaryocyte commitment.
All together, these findings elucidate novel effects of CALR mutations into the physical and functional characteristics of CALR mutant in MPNs, describing new aspects of this driver mutation during the oncogenesis of these diseases. Finally, the current data highlight the importance of studying the effects of CALR mutations during the process of megakaryocyte differentiation, as CALR mutant sub-cellular compartmentalisation and ER Ca2+ buffering activity variate during the process of megakaryopoiesis.

Citation

Morlan-mairal, M. (in press). New molecular and cellular aspects of mutant calreticulin in Myeloproliferative Neoplasms. (Thesis). University of Salford

• Thesis Type: Thesis
• Acceptance Date: Jun 29, 2018
• Deposit Date: Sep 21, 2018
• Publicly Available Date: Oct 21, 2018