Publication Date:
2018-11-29
Description:
INTRODUCTION. Calreticulin (CALR) is mutated in 20% of pts with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The most frequent mutations are a 52 bp deletion (T1) and a 5 bp insertion (T2) in exon 9, that cause a recurrent frameshift resulting in novel C-terminal sequence common to all mutant CALR proteins. Recent data indicate that interaction of mutant CALR with the thrombopoietin receptor MPL contributes to the abnormal megakaryocytopoiesis (Mk) of ET/PMF (Araki M et al & Chachoua I et al, Blood 2016; Elif S et al, Blood 2018), however full characterization of mechanisms pertaining to mutant CALR remains to be pursued. METHODS. By using CRISPR/Cas9 editing, we generated CALR knock-out (KO) variants starting from cord blood (CB) CD34+ cells, K562, UT7 and HL-60 cell lines, and CALR T1 variants from K562 and UT7 cells. Stable expression of CALR wild-type (WT), T1 and T2 was obtained by viral transfection of K562-KO cells. RESULTS. In the different genome-edited cell models, KO or T1 mutation did not result in appreciable changes in proliferation rate, cell cycle and apoptosis under standard culture conditions. However, UT7 KO and T1 cells were able to grow in the absence of GM-CSF, that was otherwise necessary for maintenance of WT counterpart, indicating cytokine independence similarly imparted by deletion or T1 mutation of CALR. To evaluate impact of mutated CALR on Mk commitment, we induced parental, KO and T1 K562 cells with phorbol-myristate acetate (PMA); at day 3 after induction, 60% and 48% of KO and T1 cells, respectively, expressed CD41/CD61 compared to 24% of parental cells (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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