Crispr/Cas9 Engineered 61bp Deletion in the Calr Gene of Mice Leads to Development of Thrombocytosis

Abstract

In a subset of patients suffering from myeloproliferative neoplasms (MPNs), calreticulin (CALR) exon 9 frameshift mutations are known to be responsible for the development of either essential thrombocythemia (ET) or primary myelofibrosis (PMF) (1, 2). The most prevalent mutations are a 52-bp deletion (del52, type-1 mutation) and a 5-bp TTGTC insertion (ins5, type-2 mutation). In these patients, the mutational status is almost always heterozygous. Our group and collaborators have recently shown that the pathogenic mutant CALR proteins require interaction with and activation of the thrombopoietin receptor (TpoR) for activation of the JAK-STAT pathway (3, 4). Until now, no knock-in mouse model of these diseases has been published. In this abstract, we show how we succeeded in creating such a model.We had shown that the murine CALR mutant proteins behave just like their human counterparts (5). Specifically, the del52, ins5 and del61 (61bp deletion, type-1) Calr mutations were able to transform Ba/F3 cells (murine pro-B lymphocytic cells normally dependent on IL-3 for growth) expressing the thrombopoietin receptor (TpoR) and render them cytokine-independent. Importantly, we also mutated the Ba/F3 genome using the widely adopted CRISPR/Cas9 system in order to create a 61-bp deletion of the exon 9 of Calr. This too successfully transformed the Ba/F3 cells, showing that endogenous levels of expression of a mutant CALR protein are sufficient to induce phenotype in vitro.Now, using the same approach, we injected C57BL/6J mouse zygotes with the same CRISPR/Cas9 constructs to create the same 61-bp deletion in the murine Calr gene. Out of 46 pups born from the procedure, one male pup was heterozygous for the 61-bp deletion. By in vitro fertilization, we subsequently obtained heterozygous Calr del61/WT pups. After inter-breeding the mice, we analyzed the blood of 12 Calr del61/WT males and 12 Calr WT/WT males (littermates) at three different timepoints (15, 18 and 22 weeks old) and found that the Calr del61/WT mice showed significantly higher levels of circulating platelets. Conversely, red blood and white blood cell numbers were the same between both groups at all time points. We further show that expression of a mutant CALR protein, in a heterozygous state, is sufficient to induce abnormal proliferation of megakaryocytes and develop an ET phenotype in vivo in mice. Follow-up in dynamics of the phenotype and bone marrow and spleen pathology (examination of myeloproliferation and fibrosis) allow comparison with the retroviral murine models of CALR-mutant MPNs and with the known features of the human disease. The only limitation of our model is the fact that the Calr del61 mutation is parentally acquired and widespread throughout the organism.With this new model, we aim to test the efficiency of various drugs to prevent or cure the MPN phenotype, such as ruxolitinib, a JAK2 type-1 inhibitor that is already used in clinics in patients suffering from CALR-mutated MPNs. We also now have a means to generate a high number of Calr del61/WT bone marrow cells to extensively study the oncogenic properties of the Calr mutations at different stages of the hematopoeisis. It will also be of great interest to study, if generated, a homozygous mutational status of Calr del61 in vivo. Thus, our system will shed light on the importance of the negatively charged tail of CALR and on the effects of the novel positively charged tail on myeloproliferation.