Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms

Abstract

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo.

Significance: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.

Figure 1.

Type I CALRdel52 mutations differentially activate the IRE1α/XBP1 pathway of the unfolded protein response (UPR). A, Left, structural comparison of wild-type (top) and mutant (bottom) calreticulin (CALR). CALR contains three distinct domains: the N-domain and P-domain are responsible for the chaperone function of the protein, although the P domain also contains one high-affinity Ca²⁺ binding site; the C-terminal domain binds Ca²⁺ with a series of acidic amino acids (denoted by blue circles), and terminates at an ER retention signal (KDEL). SP, ER signal peptide. CALR mutations occur in the C-terminal Ca²⁺ binding domain, and produce an identical 36 amino acid mutant C-terminal tail (red shaded region). The mutant C-terminus is characterized by the replacement of the acidic Ca²⁺ binding residues with positively charged residues (denoted by red circles), and loss of the KDEL sequence. SP, ER signal peptide. Right, schematic depicting C-terminal amino acid sequence of CALRwt versus type I CALRdel52 and type II CALRins5. CALRins5 retains many of the Ca²⁺ binding residues present in the wild-type protein, which are lost in the del52-mutant protein (highlighted in blue). 36 amino acid mutant C-terminal tail shared between all CALR-mutant proteins is depicted in red. B, Left, schematic depicting workflow for RNA-sequencing (RNA-seq) experiment in Ba/F3-MPL cells expressing CALR variants in MSCV-IRES-GFP (MIG) backbone. Right, GSEA plots for IRE1α-mediated UPR in Ba/F3-MPL-CALRdel52 cells versus Ba/F3-MPL-CALRwt cells (top) and Ba/F3-MPL-CALRins5 cells (bottom). C, Top, XBP1 splicing assay performed in Ba/F3-MPL cells and MPN patient cells. Top band shows the spliced form of XBP1 (s), bottom bands show the Pst1-digested unspliced form of XBP1 (us). Bottom, quantification of spliced XBP1 band. D, Western blot analysis for phospho-IRE1α, total IRE1α, and spliced XBP1 (XBP1s) in Ba/F3-MPL and UT-7-MPL cells expressing CALR variants. β-Actin was used as a loading control. E, Western blot analysis for XBP1s in CALR-expressing Ba/F3-MPL nuclear and cytosolic extracts. LSD1 was used as a nuclear marker and MEK was used as a cytosolic marker. F, qPCR for XBP1 targets DNAJB9, ADAM10, and SERP1 in UT-7-MPL cells. Each bar represents the average of three replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 2.

Type I CALRdel52 mutations lead to loss of Ca²⁺ binding function, resulting in ER Ca²⁺ depletion. A, GSEA plots for Ca²⁺ signaling pathway in Ba/F3-MPL-CALRdel52 cells versus Ba/F3-MPL-CALRwt cells (top) and Ba/F3-MPL-CALRins5 cells (bottom). B, Left, absorbance (640 nm) of 20 μg of recombinant CALRwt, CALRdel52, CALRins5 incubated with 0.025% Stains-All solution to indirectly measure the Ca²⁺ binding ability of each rCALR protein. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; **, P < 0.01). Right, Western blot analysis for calreticulin (CALR; which detects both wild-type and mutant CALR) and mutant CALR (which only detects the mutant C-terminus) for rCALR proteins used in Stains-All assay. C, Quantification of relative fluorescence of Ca²⁺ sensor in U2OS cells expressing iV2-CALRwt, CALRdel52, and CALRins5. Each bar represents the average of five individual cells. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; **, P < 0.01). D, Immunofluorescence of FLAG-tagged CALR variants (green) and ER marker calnexin (red) in U2OS cells. Nuclear staining with DAPI is shown in blue. Merge column depicts the overlay of FLAG-CALR in green with the ER in red to demonstrate colocalization (yellow). Scale bar, 10 μm.

Figure 3.

Type I mutant CALRdel52-driven ER Ca²⁺ depletion activates the IRE1α/XBP1 pathway. A, Schematic of the P+C rescue construct. The ER signal peptide was cloned just prior to the P domain to ensure proper localization of the protein. B, Immunofluorescence of empty GFP-expressing vector or P+C rescue construct in GFP-expressing backbone (green) and ER marker calnexin (red) in U2OS cells. Nuclear staining with DAPI is shown in blue. Merge column depicts the overlay of P+C in green with the ER in red to demonstrate colocalization (yellow). Scale bar, 10 μm. C, Quantification of relative fluorescence of Ca²⁺ sensor in U2OS cells expressing CALRwt + empty vector (control), CALRwt + P+C, CALRdel52 + empty vector, or CALRdel52 + P+C. Each bar represents the average of five independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; **, P < 0.01). D, Top, Western blot analysis for spliced XBP1 (XBP1s) in Ba/F3-MPL cells expressing calreticulin (CALR) variants and either empty vector (−) or P+C (+) rescue construct. β-Actin was used as a loading control. Bottom, quantification of XBP1s band relative to β-actin control from two independent Western blots (shown above and in Supplementary Fig. S3B). Analysis was performed using Thermo Fisher Scientific iBright Analysis Software. Each bar represents the average of two independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05). E, qPCR for XBP1 targets ADAM10, and SERP1 in Ba/F3-MPL cells expressing CALRwt and CALRdel52 with or without the P+C rescue construct. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01; ***, P < 0.001).

Figure 4.

Type I mutant CALRdel52-expressing cells are dependent on depleted ER Ca²⁺ to activate IRE1α/XBP1, which promotes cell survival via upregulation of BCL-2. A, Total viable cell number at 48 hours post IL3 withdrawal in Ba/F3-MPL cells expressing calreticulin (CALR) variants and either empty vector or P+C rescue construct. Ba/F3-MPL-CALRwt cells grown in the presence of IL3 was included as a control. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05). B, Top, Western blot analysis for IRE1α and XBP1s in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against IRE1α. Bottom, Western blot analysis for XBP1 in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against XBP1. C, Total viable cell number at 48 hours post IL3 withdrawal in Ba/F3-MPL cells expressing CALR variants and either a scrambled shRNA or shRNA against IRE1α (left) or XBP1 (right). Ba/F3-MPL-CALRwt cells grown in the presence of IL3 were included as a control. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01; ***, P < 0.001). D, Left, GSEA plots for BH domain binding in Ba/F3-MPL-CALRdel52 cells versus Ba/F3-MPL-CALRwt cells. Right, Heat map displaying relative expression levels of BCL-2 family genes BCL-2, BCL-xL, and MCL-1 in Ba/F3-MPL cells expressing CALR variants. E, qPCR for BCL-2 expression in Ba/F3-MPL cells (left) and UT-7-MPL cells (middle) expressing CALRwt, CALRdel52, and CALRins5, and in peripheral blood mononuclear cells (PBMC) from a healthy donor (HD) or patients with myeloproliferative neoplasms (MPN; patient number depicted below each bar) with CALRdel52 or CALRins5 mutations (right). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; ***, P < 0.001). F, Western blot analysis for BCL-2 in UT-7-MPL cells expressing CALR variants. β-Actin was used as a loading control. G, qPCR for BCL-2 expression in Ba/F3-MPL cells expressing CALR variants and either empty vector or P+C rescue construct. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). H, Left, qPCR for BCL-2 expression in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA or shRNA against XBP1. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.001). Right, Western blot analysis for BCL-2 in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA (−) or shRNA against XBP1 (+). I, Quantification of flow cytometric analysis for Annexin V/PI double positivity in Ba/F3-MPL cells expressing CALR variants and treated with or without venetoclax (1 μmol/L for 24 hours). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.001).

Figure 5.

XBP1 upregulates the IP3 receptor to induce a positive feedback loop of sustained depleted ER Ca²⁺ and IRE1α/XBP1 pathway activation in type I CALRdel52-expressing cells. A, qPCR for ITPR1 in Ba/F3-MPL cells (left) and UT-7-MPL cells (middle) expressing CALRwt, CALRdel52, and CALRins5, and in peripheral blood mononuclear cells (PBMC) from a healthy donor (HD) or patients with myeloproliferative neoplasms (MPN; patient number depicted below each bar) with CALRdel52 or CALRins5 mutations (right). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01; ***, P < 0.001). B, Western blot analysis for IP3R in Ba/F3-MPL and UT-7-MPL cells expressing calreticulin (CALR) variants. β-Actin was used as a loading control. C, qPCR for ITPR1 expression in Ba/F3-MPL cells expressing CALR variants and either empty vector or P+C rescue construct. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). D, Top, qPCR for ITPR1 expression in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA or shRNA against XBP1. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05). Bottom, Western blot analysis for IP3R in Ba/F3-MPL cells expressing CALR variants and a scramble shRNA (−) or shRNA against XBP1 (+). E, Quantification of relative fluorescence of Ca²⁺ sensor in U2OS cells expressing iV2-CALRdel52 treated with or without 2-APB (100 μmol/L for 1.5 minutes). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05). F, Top, XBP1 splicing assay performed in Ba/F3-MPL cells expressing CALR variants treated with or without 2-APB (100 μmol/L for 24 hours). Top band shows the spliced form of XBP1 (s), bottom bands show the Pst1-digested unspliced form of XBP1 (us). Bottom, quantification of spliced XBP1 band. Analysis was performed using Thermo Fisher Scientific iBright Analysis Software. G, qPCR for ADAM10 and SERP1 expression in UT-7-MPL cells expressing CALR variants treated with or without 2-APB (100 μmol/L for 24 hours). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). H, Quantification of flow cytometric analysis for Annexin V/PI double positivity in Ba/F3-MPL cells expressing CALR variants and treated with or without 2-ABP (100 μmol/L for 72 hours). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.01). I, Western blot analysis for BCL-2 in Ba/F3-MPL cells expressing CALR variants and treated with or without 2-ABP (100 μmol/L for 24 hours). β-Actin was used as a loading control.

Figure 6.

The IRE1α/XBP1 pathway represents a potential target for therapy in CALRdel52-driven MPNs. A, Western blot analysis for phospho-IRE1α, total IRE1α and XBP1s in Ba/F3-MPL cells expressing calreticulin (CALR) variants treated with or without KIRA8 (5 μmol/L for 4 hours). β-Actin was used as a loading control. B, Western blot analysis for phospho-IRE1α, total IRE1α, and XBP1s in UT-7-MPL cells expressing CALR variants treated with or without KIRA8 (5 μmol/L for 4 hours). β-Actin was used as a loading control. C, Left, total viable cell number at 72 hours post IL3 withdrawal in Ba/F3-MPL cells expressing CALR variants treated with or without KIRA8 (5 μmol/L). Right, quantification of flow cytometric analysis for Annexin V/PI double positivity in Ba/F3-MPL cells expressing CALR variants and treated with or without KIRA8 (5 μmol/L for 48 hours). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (***, P < 0.001). D, Left, total viable cell number at 72 hours post GM-CSF withdrawal in UT-7-MPL cells expressing CALR variants treated with or without KIRA8 (5 μmol/L). Right, quantification of flow cytometric analysis for Annexin V/PI double positivity in UT-7-MPL cells expressing CALR variants and treated with or without KIRA8 (5 μmol/L for 48 hours). Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). E, Relative cell viability in peripheral blood mononuclear cells from a healthy donor (HD) or patients with myeloproliferative neoplasms (MPN; patient number depicted below each bar) with CALRdel52 or CALRins5 mutations, treated with or without KIRA8 (500 nmol/L for 48 hours). Viability was determined by CellTiter-Glo assay. Each bar represents the average of three independent replicates. Error bars, SD. Significance was determined by two-tailed Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). F, Western blot analysis for BCL-2 in Ba/F3-MPL cells expressing CALR variants treated with or without KIRA8 (5 μmol/L for 24 hours). β-Actin was used as a loading control. G, Western blot analysis for IP3R in Ba/F3-MPL cells expressing CALR variants treated with or without KIRA8 (5 μmol/L for 24 hours). β-Actin was used as a loading control.

Figure 7.

Inhibition of IRE1α signaling abrogates myeloproliferative neoplasm (MPN) disease progression in vivo. A, Schematic of retroviral bone marrow transplantation assay (BMT). B, Platelet counts (PLT; left), white blood cell counts (WBC; middle), and hematocrit (HCT; right) at 16 weeks posttransplantation in the peripheral blood of recipient mice receiving CALRwt, CALRdel52, or CALRins5-expressing c-KIT⁺ BM cells treated as indicated (n = 10 in each group). Each bar represents the average of 5 mice. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). C, Histopathologic hematoxylin and eosin (H&E) sections of BM from representative CALRwt, CALRdel52, and CALRins5 mice treated with vehicle or 50 mg/kg/day KIRA8 (20× magnification, black scale bars = 100 μm). D, Megakaryocyte counts per high-power field (HPF) in BM of CALRwt, CALRdel52, or CALRins5 mice treated with vehicle or 50 mg/kg/day KIRA8. Each bar represents the average of 5 mice. Error bars, SD. Significance was determined by two-tailed Student t test (**, P < 0.01). E, IHC analysis for XBP1 in BM from representative CALRwt, CALRdel52, and CALRins5 mice treated with vehicle or 50 mg/kg/day KIRA8 (40× magnification, black scale bars = 25 μm). F, Working model: left, type I CALRdel52 proteins exhibit loss of Ca²⁺ binding function. This leads to depleted ER Ca²⁺, which in turn activates the IRE1α/XBP1 pathway. XBP1 mediates upregulation of BCL-2, which promotes cell survival, and IP3R, which facilitates continued ER Ca²⁺ efflux to sustain ER Ca²⁺ depletion and activation of IRE1α/XBP1, ultimately driving MPN. Inhibiting this pathway leads to rapid cell death in vitro and abrogation of disease progression in vivo. Right, in contrast, CALRwt and CALRins5 cells retain calreticulin (CALR)-mediated ER Ca²⁺ binding function, and thus do not hijack IRE1α/XBP1 to promote survival. Instead, canonical IRE1α/XBP1 signaling in these cells may be activated upon ER stress, wherein this pathway would initially promote adaptation and survival to restore proteostasis, but commit cells to apoptosis if stress cannot be resolved (figure created with BioRender).