Mutant calreticulin causes essential thrombocythemia

Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are categorized as myeloproliferative neoplasms (MPNs). All are characterized by the autonomous growth of one or more lineages of hematopoietic cells, splenomegaly, constitutional symptoms such as fatigue, night sweating, itching, and weight loss, and is frequently complicated by thrombosis and hemorrhage. JAK2 mutations are observed in more than 95% of PV patients, and about half of ET and PMF patients. About two-thirds of JAK2 mutationnegative ET and PMF patients harbor Calreticulin (CALR) mutations. CALR mutations with a 52-bp deletion or a 5-bp insertion in exon 9 occur in more than 80% of patients with CALR mutations, and cause frameshifts that result in proteins with novel C-terminal domains composed of many positively charged amino acids [1]. As CALR mutations are exclusively observed in conjunction with JAK2 mutations in MPN patients, mutant CALR is speculated to have a driver role in MPNs. Cytokines such as erythropoietin (EPO), thrombopoietin (TPO), and granulocyte colonystimulating factor bind to their specific cell surface receptors, commonly activate the JAK-STAT signaling cascade, and induce cytokine-dependent transient hematopoiesis [2]. In cases of JAK2 mutation, JAK2 and STAT5 are constitutively activated without cytokine stimulation, and cause autonomous cell growth (Figure 1). As CALR is best known for its endoplasmic reticulum chaperone functions, assisting in glycoprotein folding, the question has been raised as to why mutant CLAR causes MPN. We recently reported that in the presence of MPL, mutant CALR augmented the transcriptional activity of STAT5, but not of EPO receptor or CSF3R [3]. When CALR mutations are knocked-in human hematopoietic cell lines expressing MPL using the CRISPR-Cas9 system, these cell lines demonstrate increased growth or acquire cytokine-independent cell growth accompanied by STAT5 phosphorylation. These observations indicate that mutant CALR induces cytokine-independent activation of the JAK-STAT signaling cascade in cells expressing MPL, which results in increased cell growth or cytokineindependent cell growth (Figure 1). In line with these observations, CALR mutant mice demonstrate thrombocythemia and develop ET, but do not exhibit erythrocytosis or granulocytosis [3]. This is consistent with many reports in which ET patients with a CALR mutation have lower Hb levels and/or leukocyte counts compared to ET patients with a JAK2 mutation. In addition, ruxolitinib, which is a JAK inhibitor that ameliorates splenomegaly and constitutional symptoms associated with MF, and ameliorates elevated hematocrit values and splenomegaly associated with PV, attenuates the increased numbers of peripheral blood platelets and BM megakaryocytes in CALR mutant mice. The effect of ruxolitinib on mice with CALR mutations also supports the idea that mutant CALR autonomously activates the JAK-STAT signaling cascade and induces sustained thrombocytosis with increased numbers of megakaryocytes. Our observations, together with previous reports [4-7], indicate that the CALR mutation is sufficient to augment megakaryocytic cell growth and cause ET, and that CALR mutations, like the JAK2 mutation, play a driver role in MPNs. MPL is expressed not only on megakaryocytes, but also on hematopoietic stem cells (HSCs). TPO stimulation is reported to increase HSC numbers in vitro and in vivo. The proportion of HSCs in BM is higher in CALR mutant mice than in WT mice, which is probably due to the constitutional activation of STAT5 by mutant CALR in HSCs. Although the number of HSCs in BM from CALR mutant mice is elevated, HSCs with CALR mutation do not demonstrate greater self-renewal activity than WT HSCs. In the first recipients in serial competitive transplantation assays, BM cells with a CALR mutation exhibit a growth advantage compared to WT cells. In the second recipients, BM cells with a CALR mutation exhibit almost the same growth as WT BM cells. This also occurs in JAK2 Editorial