Effects of calreticulin mutations on cell transformation and immunity

Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK–STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)‐localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN‐linked mutant CRT proteins have novel frameshifted carboxy‐domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+ T cells. Mutant CRT‐linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK–STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT‐linked oncogenesis. In this review, we highlight recent findings related to the role of MPN‐linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.

with non-mutated JAK2 have mutations in exon 9 of the CRT gene (CALR). A small percentage of ET and MF patients carry mutations in the gene encoding the thrombopoietin receptor (TPOR), also known as the myeloproliferative leukemia protein (MPL). 3,4 While the most common JAK2 V617F mutation is found in around 95% of PV patients and 60% of ET and MF patients, about 15%-25% of ET and 25%-36% of MF patients carry CALR mutations, and 4%-9% of ET and MF patients carry MPL mutations. [3][4][5][6] CRT is an ER-resident calcium-binding chaperone that assists in the folding of N-glycosylated proteins. [7][8][9][10][11] Essential to this function is a glycan-binding site located in the amino-terminal domain (N-domain; Figure 1A) that interacts with asparagine-linked (Nlinked) monoglucosylated glycans of glycoproteins. 12 Over 50 CALR mutations have been linked to MPNs, with the two most frequent being a 52 base pair deletion (CRT Del52 ) and a 5 base pair insertion (CRT Ins5 ). 3,4,13 All the mutations cause a frameshift in the carboxyterminal domain (C-domain) of CRT, resulting in the creation of a novel basic C-terminus in place of the highly acidic C-terminus of the wild type (WT) protein and a loss of the C-terminal KDEL motif, 3,4 which is an endoplasmic reticulum (ER) retention sequence. This results in mutant CRT localization on the cell surface [14][15][16] and the extracellular space via Golgi-mediated secretion. 15,[17][18][19] The mechanisms by which CRT mutants drive aberrant megakaryocyte differentiation into platelets and myelofibrosis are under intense investigation. In healthy individuals, megakaryocyte growth and differentiation are triggered by the growth factor thrombopoietin (TPO) which is released from the liver. 20,21 TPO binds to its receptor (MPL or TPOR), on the surface of megakaryocytes, which leads to structural rearrangements, dimerization and downstream activation of JAK-STAT signalling cascade. [22][23][24] In MPNs, CRT mutants activate MPL signalling independently of TPO. 25,26 Binding of CRT mutants to MPL drives MPL dimerization 27 and cytokine-independent constitutive activation of the JAK-STAT pathway. 16,[25][26][27][28] The phenotypes of patients with ET caused by the JAK2 V617F mutation or the CALR mutations are different. These include markedly increased platelet counts in ET patients with CALR mutations compared to those with JAK2 mutations, but a relatively lower thrombotic risk. 3,4,29 Based on the unique clinical aspects of mutant CRT-mediated MPNs and the distinct interactions that trigger cell transformation, a better understanding of the role of mutated CRT in the pathogenesis of MPNs could lead to new diagnostics and treatments for MPN patients with CALR mutations.
Various mouse models have been developed which recapitulate several aspects of CALR mutation-driven MPN disease. 26,[30][31][32][33][34][35][36] These include an increase in the blood platelet counts (thrombocytosis) and megakaryocyte counts in the BM. 26,[30][31][32][33][34][35][36] The expression of CRT Del52 in a homozygous context in mice drives more severe thrombocytosis when compared to either the heterozygous CRT Del52 mice 32,34 or the CRT Ins5 mice. 34 MF was observed only in CRT Del52 -based mouse models 30,32,34 but not in CRT Ins5 expressing mice. 30,34 Homozygous CRT Del52 mice show minor or mild fibrosis in the BM and spleen accompanied by splenomegaly, decrease in the BM cellularity, megakaryocyte hyperplasia and extramedullary hematopoiesis. 32,34 Heterozygous CRT Del52 knock-in mice do not progress to MF, in contrast to MPN patients heterozygous for CRT Del52 who often develop MF. [32][33][34] This could be explained by reduced activation of murine MPL signalling compared to human MPL activation by either murine or human CRT mutants. 33,34 Some studies have reported that mice expressing mutant CRT proteins exhibit the increased self-renewal capacity of HSCs when compared to WT HSCs 30,33,34 but this has not been observed in other studies. 31,32 The discrepancies between the different mouse F I G U R E 1 Structural models of CRT Del52 monomer (A) and dimer (B). The CRT monomer structure was generated by AlphaFold. 90 The dimer was modelled using the crystal structure of human CRT D71K 91 (PDB ID: 5LK5, subunits G and E) as described earlier. 52 Residues forming ionic pairs at the dimerization interfaces (K142, R162, D165) are shown as blue and green sticks for the two subunits. Residues forming the glycan binding site (C105, Y109, K111, Y128 and G133) 12 are shown as purple sticks. Residues suggested to be involved in Zn 2+binding (H99-C163, H145-D135 and H170-D166) 54 are shown as cyan sticks. A Ca 2+ ion in the high-affinity site of each monomer is shown as a red sphere. Figures 1, 2, 4 and 5 were prepared using the PyMOL Molecular Graphics System (version 1.8.4.2) Schrödinger, LLC. models could be related to specific knock-in constructs used or to the specific type of mouse model, which can affect the expression levels, the expression patterns, the relative functionality of the mutant CRT proteins, and whether or not endogenous calreticulin is also expressed. A recent study shows that CRT haploinsufficiency itself augments the self-renewal activity of HSCs in mice 35 which is of relevance to human disease, where the majority of CALR mutations occur in a heterozygous context. 3,4 Although, the different mouse models clearly substantiate the role of MPN-linked CRT mutations as drivers of MPNs, there are differences not only between the different models but also between the MPN characteristics reported in mouse models compared to human MPNs. For instance, in contrast to the observations in mouse models, 30,34,36 CRT Ins5 expressing ET patients have higher platelet counts compared to CRT Del52 ET patients. 37,38 The different mouse models that are described and their recapitulation of human disease has recently been extensively reviewed elsewhere 39 which can be referred to for more detailed insights.

| C ALCI UM B IND ING BY C ALRE TI CULIN AND ITS RELE VAN CE TO C AN CER
CRT is a 46 kDa protein composed of a conserved globular Nterminal lectin domain, a flexible proline-rich polypeptide-binding P-loop, and a C-terminal domain ( Figure 1A). An important function of CRT is the buffering of calcium (Ca 2+ ) ions in the ER, via multiple low affinity binding sites for Ca 2+ ions, 40,41 which play a critical role in the maintenance of ER Ca 2+ homeostasis. In addition to the high-affinity Ca 2+ binding site at D328 of the N-domain (K D ~ 20 μM) ( Figure 1A), 4-6 low-affinity, high-capacity Ca 2+ binding sites (K D ~ 600 μM) were identified in the C-terminal region of CRT. 41 Ca 2+ binding to these sites increases the structural stability of the C-domain of CRT 41 which can undergo Ca 2+ -dependent unfolding or folding into an α-helix and contact with the flexible P-loop. 42 Maintenance of Ca 2+ levels in the ER via sequestration by Ca 2+ binding proteins is important for many calcium-dependent proteins and processes. 43 Low calcium levels can lead to disruptions of homeostasis in the ER and activation of the unfolded protein response (UPR), leading to adjustments to protein folding in the ER to ensure cell survival. 44 Although both the CRT Del52 and CRT Ins5 mutants have novel C-terminal domains, a recent study by Ibarra et al. indicates that CRT Ins5 and CRT Del52 differ in their calcium binding properties and the downstream pathogenic effects. 36 Compared with CRT Ins5 , CRT Del52 is suggested to lose more of its calcium binding capacity due to the smaller number of acidic residues that are retained in the novel C-domain of CRT Del52 . 36 Restoration of the calcium-binding capacity in CRT Del52 cells via co-expression of just the P + C domains of WT CRT decreased the survival of CRT Del52 cells, suggesting that CRT Del52 requires a low ER Ca 2+ environment (high cytosolic calcium) to maintain cell survival. 36 Megakaryocytes that express CRT Del52 display higher levels of Ca 2+ mobilization into the cytosol compared to cells obtained from patients with a JAK2 mutation or a CRT Ins5 mutation. 38 Ibarra et al. further demonstrated that, compared to WT CRT and CRT Ins5 expressing cells, CRT Del52 expressing cells exhibited an upregulation of genes related to the inositol requiring-enzyme 1 alpha (IRE1α)/X-box-binding protein 1(XBP1) UPR pathway. This, in turn, stimulated the expression of B-cell lymphoma (BCL)-2 protein, 36 an anti-apoptotic protein that prevents cell death and increases cell survival. 45 It was also seen that CRT Del52 cells were able to sustain this IRE1α/XBP1 response by increasing the transcription of the ITPR1 gene that encodes the Inositol-1,4,5-triphosphate (IP3R) receptor, one of the mediators of calcium efflux from the ER.
CRT Del52 was thus able to maintain consistent depletion of Ca 2+ levels in the ER, 36 and a constitutive store-activated Ca 2+ entry (SOCE) response. 46 These studies suggest an important distinction between CRT Del52 and CRT Ins5 expressing cells; CRT Del52 expressing cells promote cell survival via activation of UPR which is not seen in CRT Ins5 cells. Further research can be conducted to examine the distinct oncogenic pathways used by CRT Del52 and CRT Ins5 , which could lead to therapeutics based on the specific mechanisms of pathogenesis.
Megakaryocytes from MPN patients with CALR mutations also display spontaneous calcium influx into the cytosol via the calcium release-activated calcium channel protein 1 (ORAI1), 46 residing in the plasma membrane (PM), which mediates the SOCE response. 47 Upon Ca 2+ depletion in the ER, the stromal interaction molecule 1 (STIM1), a calcium sensor located in the ER membrane, undergoes conformational changes, which weakens its interaction with ERp57, 48 a thiol-disulfide oxidoreductase. ERp57 is a co-chaperone for WT CRT 49 that is shown to be impaired in binding to CRT mutants. 50 Dissociation of the complex between STIM1, ERp57 and CRT mutants in the ER, is suggested to result in enhanced STIM1 oligomerization and relocation to ER-PM junctions, where it gates the ORAI1 channel, 47 thus promoting enhanced SOCE activation, Ca 2+ influx into the cytosol and cell proliferation. 46 Inhibition of SOCE reduced the cytokine-independent proliferation of mutant CRT-expressing megakaryocytes. 46 Overall, altered cellular calcium homeostasis is a component of the pathogenic effects of the CRT mutants, and some studies suggest that disruptions induced by CRT Del52 are more severe than those induced by CRT Ins5 .

| MUTANT CRT MULTIMERIZ ATION
Compared to WT CRT, CRT Del52 and CRT Ins5 have a high propensity to form homomultimers. 51,52 Cytokine-independent MPL activation requires homomultimerization of CRT mutants which is dependent on the intermolecular interactions between mutant CRT monomers involving the novel C-domains [51][52][53] and/or the non-mutated N-domains. 52,54 The novel C-domain cysteines (C400 and C404) along with the C163 residue in the N-domain mediate the formation of disulfidelinked dimers/multimers of CRT Del52 . 52 Alanine substitutions of these three cysteines (CRT Del52 -3CA ) abrogated the disulfide-linked dimerization of CRT Del52 and reduced its ability to bind MPL. However, the CRT Del52 -3CA mutant exhibited only a small reduction in the MPL-mediated cell proliferation Combination of CRT Del52 -3CA with mutations of N-domain residues, specifically, D165, D166 and H170, further reduced the dimer formation, accompanied by a significant impairment of MPL binding and cytokine-independent proliferation. 52 Oligomerization was also significantly decreased upon truncation of the novel C-domain of CRT Del52 , especially for CRT  in which 36 C-terminal residues are deleted. 52  Another recently proposed model of CRT Del52 dimerization is based on experimental and computational results. 53 The first 28 residues (367-394) of the CRT Del52 C-tail were experimentally determined to be important for CRT Del52 and MPL dimerization as well as for cytokine-independent cell proliferation. 53 In this model, the two CRT Del52 monomers dimerize through coiled-coil interactions between the positively charged C-terminal α-helices (residues 367-391) while the cysteines of the CRT Del52 C-terminus are not involved in the formation of productive dimers. 53 Additional structural studies are needed to fully uncover the molecular structures of functionally relevant dimers of CRT mutants. A model that could resolve some of the discrepancies is further discussed below.

| MPL AC TIVATI ON AND S I G NALLING
MPL is a cell surface class I cytokine receptor that regulates megakaryocyte differentiation and platelet production. MPL is synthesized and folded within the ER via the calreticulin/calnexin cycle.  Figure 2A; further discussed below). The membrane distal CRM1 acts as a brake on MPL activation and cell proliferation, 55 and is required for TPO binding. 55,56 Mutations of residues located in the hinge between the D1 and D2 (F104, F45, L103, D261 and L265) impaired TPO binding and/or TPO-dependent MPL activation ( Figure 2B; further discussed below). 56,57 MPL activation is driven by dimerization of TMDs in a specific helical orientation with S505 at the contact interface. 23,58 In addition to the inactive and the physiologically active conformations of the TMD dimers, five other dimeric orientations of murine MPL transmembrane helices were identified, which induced cell proliferation and in vivo myeloproliferative disorders. 58 A juxtamembrane motif 514 KWQFP 518 in the ICD of murine MPL regulates the transmembrane helix orientation and prevents receptor self-activation. [58][59][60] Furthermore, Box1 ( 530 PSLPDL 535 ) and Box2  The TM α-helical dimer was generated by the TMDOCK server 93 to substitute for the AFM-generated TMD dimer. The TMDOCK-modeled TMD dimer has a left-handed helix arrangement with V501 and S505 at the dimerization interface, which corresponds to the active receptor state. 23,58 MPL subunits (coloured green and cyan) and TPO (coloured purple) are shown by cartoon and surface representations. N-linked glycans attached to N117, N178, N298 and N358 of each MPL subunit are shown as red sticks. The WGSWS and WSSWS motif in D2 and D4, respectively, are shown by sticks coloured dark blue. Cysteine residues of MPL and TPO are shown as orange spheres. Mutated residues associated with the constitutive activation of MPL (V501, S505 and W515) 62-64 are marked by red spheres. The inset in B shows residues of two MPL subunits (F45, E46, D47, L103, F104, D261 and L265) contacting two sides of the TPO fourα-bundle that are shown to be important for TPO binding and/or TPO-induced MPL activation. 56,57

| MUTANT CRT-MPL COMPLE XE S AND DYS REG UL ATED MPL S I G NALLING IN MPN S
CRT Del52 and CRT Ins5 induce cytokine-independent cell proliferation and oncogenesis via specific interaction with MPL, 16 of CRT Del52 , 52,53 as the deletion of 28 (∆28) and 36(∆36), but not of 19 (∆19) residues from the CRT Del52 C-terminus reduced the ability of CRT Del52 to induce cytokine-independent proliferation of MPLexpressing cells. 52 The ECD of MPL has four N-glycosylation sites, N117, N178, N298 and N358. Removal of N117 site alone, as well as the mutations of any three MPL glycosylation sites, lowered the surface expression of MPL and TPO-dependent cell proliferation. 77 N117 is essential for activation of MPL signalling by CRT Del52 and CRT Ins5 , while N178 supports weak MPL activation by CRT Ins5 . 25 CRT mutants determine the transport of partially immature MPL (with N117linked mannose-rich glycan 27 ) from ER to the cell surface via the secretory pathway. 25,27 When complexes of MPL with CRT mutants are trafficked through the Golgi apparatus, N178, N298 and N358 achieve mature glycosylation, whereas N117 is protected from maturation by binding to mutant CRT. 27 CRT mutants also rescue traffic to the cell surface and activation of traffic-defective MPLs, including K39N, R102P, G509N mutants and constructs carrying ER-retention signals. 27 Although MPL and mutant CRTs become engaged in the ER, surface localization of the mutant CRT-MPL complex is indispensable for the activation of MPL signalling. 14 CRT Del52 interacts with D1 of MPL not only by binding the N117linked glycan but also by forming ionic interactions between the basic residues in the CRT Del52 C-tail (residues 378-391) and negatively charged patches centred at 44 TFED 47 and 52 WDEE 55 in MPL ECDs. 53 A patch of 8 hydrophobic ( 104 FFPLHLWV) residues was identified in the hinge between D1-D2 subdomains of MPL that is required for MPL activation by CRT mutants but not for binding. 27 CRT mutants are also detected in the plasma of MPN patients. 15,19,52 A recent study shows that the secreted forms of CRT mutants are stabilized by binding to the soluble transferrin receptor 1 (sTFRC). 15 Paracrine activation of MPL signalling by secreted forms of CRT mutants was not observed when healthy cells expressing MPL and WT CRT were treated with supernatants from CRT Del52 expressing cells. 18 However, a recent study demonstrates that exogenous recombinant CRT Del52 promotes cytokine-independent proliferation, particularly of cells expressing both mutant CRT and MPL. 15 Cells expressing both MPL and mutant CRTs expose immature MPL glycans on the cell surface to induce additional activation by exogenously supplied CRT mutants. 15 Despite the accumulation of a vast amount of experimental data supporting the formation of MPL-CRT complexes, the exact mode of binding of CRT mutants to MPL and the molecular mechanism of mutant CRT-induced MPL activation remains to be characterized.
Modelling with AFM allowed us to construct tentative structural models for different conformations of the CRT Del52 :MPL (2:2) heterotetrameric complex (Figure 4) that has been suggested based on size exclusion chromatography data. 27 The previously proposed CRT Del52 dimer ( Figure 1B) can be unambiguously docked onto the AFMgenerated closed conformations of MPL dimers due to the matching of distances between N117 of two MPL subunits and between K111 residues in the two glycan binding sites of the CRT Del52 dimer ( Figure 4A). In this model, the small α-helical fragments (residues 400-406) of the CRT Del52 C-tail can interact with hydrophobic residues from the MPL ligand-binding pocket, while basic C-tail residues from the 385 RKMR 388 fragment are in close proximity to residues 52 WDEE 55 forming a negative patch in MPL implicated in the binding of mutant CRT. 53 However, the CRT Del52 dimer appears to be incom-

| MH C CL A SS I ANTI G EN PRE S ENTATI ON AND T CELL AC TIVATI ON BY MUTANT CRT IN MPN
CRT is a component of a membrane-bound multi-protein machinery in the ER called the MHC class I peptide loading complex (PLC). 78 The PLC assists in the assembly of MHC class I molecules with peptides to allow CD8 + T cells to distinguish healthy and compromised cells. Within the PLC, there are two "editing" modules, each containing a peptide-free MHC class I heavy chain in complex with β2- Interactions of N-linked glycan (shown by red sticks) attached to N86 of MHC class I heavy chain (coloured blue) with calreticulin (coloured yellow). MHC class I heavy chain and calreticulin molecules that were extracted from the cryo-EM structure of the human PLC (PDB ID: 7qpd, subunits M and C) 80 are shown by cartoon and surface representations coloured blue and yellow, respectively. Residues from the glycan binding pocket of calreticulin 54 are shown as green spheres. by CRT mutants is undoubtedly a key mechanism driving aberrant megakaryocyte growth, platelet production and myelofibrosis in ET and MF patients carrying CALR mutations. Understanding the structural details of complexes formed between CRT mutants, MPL and JAK2 proteins will allow for the design of inhibitors that could specifically target these complexes. Moreover, MPN-linked CRT mutants alter cellular calcium homeostasis, which likely contributes to cellular survival and proliferative advantages. Uncovering the differ-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm that there are no conflicts of interest.