Disease Phenotype and Clonal Amplification in Calreticulin del52 and ins5 Knock-in Mice Are Dependent on the Type of Mutations and Gene Dosage

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.311-311
• Main Authors: Benlabiod, Camélia, da Costa Cacemiro, Maira, Balligand, Thomas, Panneau-Schmaltz, Barbara, Muller, Delphine, Touchard, Laure, Gonin, Patrick, Villeval, Jean-Luc, Constantinescu, Stefan N, Raslova, Hana, Vainchenker, William, Plo, Isabelle, Marty, Caroline
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-127918

Introduction BCR-ABL-negative myeloproliferative neoplasms (MPNs) result from the transformation of a hematopoietic stem cell (HSC). Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutations induce a frameshift to the same alternative reading frame generating a new C-terminal tail. The two most frequent CALR mutations are a 52 bp deletion (del52) or type 1 and a 5 bp insertion (ins5) or type 2. In patients, del52 and ins5 are equally found in ET but del52 is more frequent in PMF. In mouse retroviral model, del52 mice progress from ET to myelofibrosis (MF) while ins5 mice remain mostly with an ET. Methods In order to study the effect of endogenous levels of del52 and ins5 in hematopoiesis, we generated conditional knock-in (KI) mice expressing the murine CALR del52 or ins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase (Scl-CreERT). We have also used Ubi-GFP transgenic mice to perform competitive engraftments. Results After tamoxifen-induction, both del52 and ins5 KI mice developed a rapid thrombocytosis, more severe in the homozygous than the heterozygous setting. In contrast, leukocytosis was observed only in homozygous setting. At similar zygosity, del52 induced a higher thrombocytosis compared to ins5. After 10 months of induction, both the bone marrow (BM) and the spleen of homozygous del52 KI mice and, to a much lower extent of homozygous ins5 KI mice, presented a significant increase in megakaryocytes (MKs) and in MK progenitors by flow cytometry. Von Willebrand factor staining showed that both del52 and ins5 homozygous mice displayed giant polylobulated MKs, associated with a similar increase in ploidy (mean ploidy 32N-33N). Heterozygous del52 presented also an increase ploidy of MK (mean of 25N) compared to controls (mean of 17N), whereas the MK ploidy of heterozygous ins5 mice was similar to control mice. The increase in number and size of MKs in homozygous del52 mice partially explained the significant decrease in BM cellularity and the splenomegaly. Moreover, we observed a decrease in BM erythroblasts and, in spleen, an increase in both erythroblast and granulocytic precursors together with a decrease in lymphocytes associated with a major disorganization of white pulp territories. Thus, the del52 homozygous KI mice developed features of a MF-like disease further illustr