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Poster display session

4585 - CALR mutations and their link with cellular calcium during megakaryocyte hyperplasia in MPNs

Date

11 Sep 2017

Session

Poster display session

Presenters

Maria Morlan Mairal

Citation

Annals of Oncology (2017) 28 (suppl_5): v1-v21. 10.1093/annonc/mdx361

Authors

M. Morlan Mairal1, P. Papadopoulos2, J. Hetmanski3, P.T. Caswell3, M. Krstic-Demonacos1, A. Aziz1

Author affiliations

  • 1 Environment And Life Sciences, University of Salford, M5 4WT - Salford/GB
  • 2 Dept Of Hematology, Hospital Clinico San Carlos, 28040 - Madrid/ES
  • 3 Wellcome Trust Centre For Cell-matrix Research, University of Manchester, M13 9PT - Manchester/GB
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Resources

Abstract 4585

Background

Megakaryocyte hyperplasia is a major characteristic of two myeloproliferative neoplasms (MPNs) known as essential thrombocythemia (ET) and Primary myelofibrosis (PMF). About 35% of ET and PMF patients harbour somatic calreticulin (CALR) mutations. CALR is a calcium (Ca2+) buffering protein within the endoplasmic reticulum (ER). Ca2+ is an important element for megakaryocyte functions; however the impact of CALR mutations in cellular Ca2+ during megakaryocyte hyperplasia remains elusive.

Methods

I-TASSER software was used to study the aminoacid charge using the 3D structure of CALR mutant. Marimo cells and overexpressing CALR mutant HEK293T and DAMI cells were used as cellular models. CALR cellular localization was addressed by flow cytometry and confocal microscopy. Basal Ca2+ levels were measured by Fluo-8 staining. Furthermore cells were treated with Fendiline and BTP-2 calcium channel blockers to manipulate cellular Ca2+.

Results

The present study shows that CALR mutations change the aminoacid charge of the Ca2+ binding region of CALR and that mutant CALR is localized outside the ER, within the cytoplasm and the cellular membrane. These results suggest that CALR mutations could be affecting the Ca2+ buffering activity within the ER. Therefore, we further analysed Ca2+ basal levels in CALR mutant cells, and our results showed that CALR mutations show lower cellular Ca2+levels. These results lead us to think that low intracellular Ca2+ levels could be the driving force of megakaryocyte hyperplasia characteristic of ET and PMF. Therefore, we induced low intracellular Ca2+ levels in leukemic blast by using Ca2+ channel blockers and our results showed that treated cells display an increase of the megakaryocyte marker CD41a in the cell surface, suggesting an induction of megakaryopoiesis in these cells.

Conclusions

These findings elucidate that low intracellular Ca2+ caused by CALR mutations could be the driving force of megakaryocyte formation in ET and PMF. This study shows the relevance to understand the role of cellular calcium during megakaryocyte formation and this could unravel the pathogeny of CALR mutant in MPNs.

Clinical trial identification

Legal entity responsible for the study

University of Salford

Funding

University of Salford

Disclosure

All authors have declared no conflicts of interest.

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