Homomultimerization of mutant calreticulin is a prerequisite for MPL binding and activation

Calreticulin is an ER (endoplasmic reticulum) luminal Ca2+-buffering chaperone. The protein is involved in regulation of intracellular Ca2+ homoeostasis and ER Ca2+ capacity. The protein impacts on store-operated Ca2+ influx and influences Ca2+-dependent transcriptional pathways during embryonic development. Calreticulin is also involved in the folding of newly synthesized proteins and glycoproteins and, together with calnexin (an integral ER membrane chaperone similar to calreticulin) and ERp57 [ER protein of 57 kDa; a PDI (protein disulfide-isomerase)-like ER-resident protein], constitutes the 'calreticulin/calnexin cycle' that is responsible for folding and quality control of newly synthesized glycoproteins. In recent years, calreticulin has been implicated to play a role in many biological systems, including functions inside and outside the ER, indicating that the protein is a multi-process molecule. Regulation of Ca2+ homoeostasis and ER Ca2+ buffering by calreticulin might be the key to explain its multi-process property.

The genetic landscape of classical myeloproliferative neoplasm (MPN) is in large part elucidated. The MPN-restricted driver mutations, including those in JAK2, calreticulin (CALR), and myeloproliferative leukemia virus (MPL), abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors, more particularly the STATs. The most frequent mutation, JAK2V617F, activates the 3 main myeloid cytokine receptors (erythropoietin receptor, granulocyte colony-stimulating factor receptor, and MPL) whereas CALR or MPL mutants are restricted to MPL activation. This explains why JAK2V617F is associated with polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis (PMF) whereas CALR and MPL mutants are found in ET and PMF. Other mutations in genes involved in epigenetic regulation, splicing, and signaling cooperate with the 3 MPN drivers and play a key role in the PMF pathogenesis. Mutations in epigenetic regulators TET2 and DNMT3A are involved in disease initiation and may precede the acquisition of JAK2V617F. Other mutations in epigenetic regulators such as EZH2 and ASXL1 also play a role in disease initiation and disease progression. Mutations in the splicing machinery are predominantly found in PMF and are implicated in the development of anemia or pancytopenia. Both heterogeneity of classical MPNs and prognosis are determined by a specific genomic landscape, that is, type of MPN driver mutations, association with other mutations, and their order of acquisition. However, factors other than somatic mutations play an important role in disease initiation as well as disease progression such as germ line predisposition, inflammation, and aging. Delineation of these environmental factors will be important to better understand the precise pathogenesis of MPN.

Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.