Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage

Abstract

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALR mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALR mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALR HSCs were more proliferative in vitro, but neither CALR nor CALR displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF.

Essential thrombocythaemia with mutation inMPL: clinicopathological correlation and comparison withJAK2V617F-mutated andCALR-mutated genotypes

“…To this respect, a striking finding of the present work was the absence of histological features of myelofibrosis in patients with MPL -mutated ET, which contrasts with the frequent presence of this finding in CALR -mutated ET, with 11% of such patients being reclassified as actually having prefibrotic myelofibrosis. In this regard, it has been recently described that CALR del/+ mice develop a transplantable ET-like disease whereas homozygous CALR del/del mice develop extreme thrombocytosis accompanied by features of myelofibrosis 20. In this sense, the higher frequency of histological findings of myelofibrosis in patients with CALR -mutated ET provides further evidence in support of a molecular subclassification of MPN instead of one relying only on clinical and morphological phenotype.…”

Mechanism underlying the development of myeloproliferative neoplasms through mutant calreticulin

“…Retrovirus transduction of human CALR del52 and ins5 into lineage‐negative or c‐KIT + mouse bone marrow cells, and subsequent transplantation to lethally irradiated mice, induced an increase in platelet count, one of the features of ET 6,7 . An increase in platelets was also observed in a transgenic mouse harboring human CALR del52 driven by H‐2Kb promoter, 8 or in a knock‐in mouse conditionally expressing CALR del52 by endogenous promoter 9 . In the mouse transplanted with CALR del52 cells, the number of red and white blood cells was reduced later, along with an increase in spleen weight and fibrosis in both bone marrow and spleen, thus showing the phenotype of secondary myelofibrosis, and indicating the potential of mutant CALR in the development of bone marrow fibrosis observed in PMF.…”

Progress in elucidation of molecular pathophysiology of myeloproliferative neoplasms and its application to therapeutic decisions

“…Intriguingly, only the CRISPR-Cas9-generated germline Calr del52 mice show competitive advantage of mutant clones over wild-type clones in the transplantation assay [83]. Neither Shida nor Li's mice model shows such phenomenon [80,81].…”

“…Several mutant Calr mice models have also been established, all exhibit an ET-like phenotype [80][81][82][83]. Myelofibrosis phenotype was observed only in the homozygous Calr mutant mice model [81]. The germline homozygous Calr del52 mice are embryonic lethal due to developmental deficiency in the ventricular walls [83].…”