Contributions
Abstract: S884
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:45 - 17:00
Location: Room A8
Background
The acquisition of somatic mutations in the hematopoietic stem cell (HSC) compartment leading to the constitutive activation of the JAK-STAT signaling pathway drives the development of myeloproliferative neoplasms (MPNs). Besides JAK2V617F, recurrent genetic abnormalities in the exon 9 of calreticulin (CALR) gene are the second most frequent driver mutations found in essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutants induce a +1-bp frameshift of the same alternative reading frame and generate a common novel C-terminus tail. Retroviral models have shown that these mutants are driver of a MPN phenotype in mice dependent on the thrombopoietin receptor.
Aims
We investigated the consequences of physiological expression of one of the most frequent CALR mutations, the 52-bp deletion (del52) or type 1, in the pathophysiology of MPNs.
Methods
We generated a knock-in (KI) mouse model. As the analogous 52-bp deletion found in human patients would generate a mutated C-terminus tail with a different amino acid sequence in mice, our approach was to introduce fused mouse exons 8 and 9 framed with loxP sites and to humanized the C-terminal mutant tail in mouse exon 9. Floxed KI mice were crossed with tamoxifen-inducible Scl-CreERT (and Vav-Cre) mice to generate murine calr with the human mutated C-terminus sequence. Blood parameters and the different bone marrow and spleen hematopoietic compartments were analyzed over one year.
Results
Both heterozygous and homozygous KI del52 mice were produced and developed a del52 dose-dependent increase in both platelet and white blood cell counts. In contrast to heterozygous mice, ET-like phenotype in the homozygous del52 mice progressed towards mild myelofibrosis in bone marrow (BM) but not in spleen after one year of induction. Myelofibrosis was accompanied with a decrease BM cellularity and splenomegaly. Both BM and spleen from heterozygous and homozygous mice presented megakaryocytic (MK) hyperplasia revealed using von willebrand factor staining and flow cytometry. In vivo, MKs achieved significantly higher ploidy in KI mice than in WT littermates (modal ploidy of 32N compared to 16N in WT). In BM, MK frequency increased mostly at the expense of erythroid (CD71+Ter-119+) cells. In spleen, increased MK frequency was associated with an increased frequency of erythroid cells resulting in a decreased frequency of B (B220+) and T (CD3+) cells. We observed a del52 dose-dependent increase in the frequency of immature progenitors (SLAM cells) and MK progenitors (MkP) both in BM and spleen as well as a thrombopoietin-independent growth of the MK progenitors (CFU-MKs) in BM. Finally, using competitive BM transplantation with different ratios of homozygous del52 KI cells and WT GFP+ cells we found that the homozygous del52 BM cells were able to outcompete WT BM cells and induce thrombocytosis from an initial 25% competitive engraftment.
Conclusion
Altogether, these results demonstrate that CALR del52 is sufficient to dose-dependently induce a thrombocytosis progressing to myelofibrosis in KI mice, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPNs or cooperation with others mutated molecules.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Essential Thrombocytemia, Megakaryocyte, Myelofibrosis, Myeloproliferative disorder
Abstract: S884
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 16:45 - 17:00
Location: Room A8
Background
The acquisition of somatic mutations in the hematopoietic stem cell (HSC) compartment leading to the constitutive activation of the JAK-STAT signaling pathway drives the development of myeloproliferative neoplasms (MPNs). Besides JAK2V617F, recurrent genetic abnormalities in the exon 9 of calreticulin (CALR) gene are the second most frequent driver mutations found in essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutants induce a +1-bp frameshift of the same alternative reading frame and generate a common novel C-terminus tail. Retroviral models have shown that these mutants are driver of a MPN phenotype in mice dependent on the thrombopoietin receptor.
Aims
We investigated the consequences of physiological expression of one of the most frequent CALR mutations, the 52-bp deletion (del52) or type 1, in the pathophysiology of MPNs.
Methods
We generated a knock-in (KI) mouse model. As the analogous 52-bp deletion found in human patients would generate a mutated C-terminus tail with a different amino acid sequence in mice, our approach was to introduce fused mouse exons 8 and 9 framed with loxP sites and to humanized the C-terminal mutant tail in mouse exon 9. Floxed KI mice were crossed with tamoxifen-inducible Scl-CreERT (and Vav-Cre) mice to generate murine calr with the human mutated C-terminus sequence. Blood parameters and the different bone marrow and spleen hematopoietic compartments were analyzed over one year.
Results
Both heterozygous and homozygous KI del52 mice were produced and developed a del52 dose-dependent increase in both platelet and white blood cell counts. In contrast to heterozygous mice, ET-like phenotype in the homozygous del52 mice progressed towards mild myelofibrosis in bone marrow (BM) but not in spleen after one year of induction. Myelofibrosis was accompanied with a decrease BM cellularity and splenomegaly. Both BM and spleen from heterozygous and homozygous mice presented megakaryocytic (MK) hyperplasia revealed using von willebrand factor staining and flow cytometry. In vivo, MKs achieved significantly higher ploidy in KI mice than in WT littermates (modal ploidy of 32N compared to 16N in WT). In BM, MK frequency increased mostly at the expense of erythroid (CD71+Ter-119+) cells. In spleen, increased MK frequency was associated with an increased frequency of erythroid cells resulting in a decreased frequency of B (B220+) and T (CD3+) cells. We observed a del52 dose-dependent increase in the frequency of immature progenitors (SLAM cells) and MK progenitors (MkP) both in BM and spleen as well as a thrombopoietin-independent growth of the MK progenitors (CFU-MKs) in BM. Finally, using competitive BM transplantation with different ratios of homozygous del52 KI cells and WT GFP+ cells we found that the homozygous del52 BM cells were able to outcompete WT BM cells and induce thrombocytosis from an initial 25% competitive engraftment.
Conclusion
Altogether, these results demonstrate that CALR del52 is sufficient to dose-dependently induce a thrombocytosis progressing to myelofibrosis in KI mice, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPNs or cooperation with others mutated molecules.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Essential Thrombocytemia, Megakaryocyte, Myelofibrosis, Myeloproliferative disorder