Skip to main content

Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice

Abstract

Frameshifts in the Calreticulin (CALR) exon 9 provide a recurrent driver mutation of essential thrombocythemia (ET) and primary myelofibrosis among myeloproliferative neoplasms (MPNs). Here, we generated knock-in mice with murine Calr exon 9 mimicking the human CALR mutations, using the CRISPR-Cas9 method. Knock-in mice with del10 [Calrdel10/WT (wild−type) mice] exhibited an ET phenotype with increases of peripheral blood (PB) platelets and leukocytes, and accumulation of megakaryocytes in bone marrow (BM), while those with ins2 (Calrins2/WT mice) showed a slight splenic enlargement. Phosphorylated STAT3 (pSTAT3) was upregulated in BM cells of both knock-in mice. In BM transplantation (BMT) recipients from Calrdel10/WT mice, although PB cell counts were not different from those in BMT recipients from CalrWT/WT mice, Calrdel10/WT BM-derived macrophages exhibited elevations of pSTAT3 and Endothelin-1 levels. Strikingly, BMT recipients from Calrdel10/WT mice developed more severe pulmonary hypertension (PH)—which often arises as a comorbidity in patients with MPNs—than BMT recipients from CalrWT/WT mice, with pulmonary arterial remodeling accompanied by an accumulation of donor-derived macrophages in response to chronic hypoxia. In conclusion, our murine model with the frameshifted murine Calr presented an ET phenotype analogous to human MPNs in molecular mechanisms and cardiovascular complications such as PH.

To the editor,

CALR frameshifts provide a recurrent myeloproliferative neoplasm (MPN) driver [1]. Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disease characterized by increased pulmonary arterial (PA) pressure. Bone marrow (BM)-derived cells and perivascular inflammatory infiltrates contribute to PA remodeling in PH [2, 3]. Among 5 etiological groups, the WHO group-V PH encompasses multifactorial mechanisms, including MPNs, which are often complicated by PH, with 5%-60% of the prevalence [4,5,6]. MPN-related PH is associated with crucial features, such as thromboembolism and hypermetabolic state [5]. However, the association of PH with CALR mutation remains uncertain. Here, we generated Calrdel10/WT and Calrins2/WT knock-in mice (Fig. 1a, Additional file 1: Fig. S1), investigated their hematopoiesis, and clarified the role of hematopoietic Calr mutation in PH using BM transplantation (BMT) and chronic hypoxia, which provokes PH [7].

Fig. 1
figure1

Hematopoietic cells with Calr mutation exacerbate the development of pulmonary hypertension in response to chronic hypoxia. a The knock-in mice with C57BL/6 J background carrying frameshifted murine Calr, del10 (Calrdel10/WT mice) and ins2 (Calrins2/WT mice) were generated using the CRISPR-Cas9 method. Structure of wild-type (WT) and frameshifted murine CALR proteins are shown. Both generated mutant proteins with shortened calcium-buffering sites and absent KDEL sequence, which is the signal to retain the CALR protein in the endoplasmic reticulum. b Leukocyte (white blood cell) counts (WBC), red blood cell counts (RBC), and platelet counts (PLT) in WT mice (CalrWT/WT mice, n = 21), Calrins2/WT mice (n = 17), and Calrdel10/WT mice (n = 16) in the peripheral blood. *P < 0.05 versus the WT group. c Schematic diagram of the experimental design of bone marrow (BM) transplantation (BMT). BM cells from control CalrWT/WT mice or Calrdel10/WT mice were injected into the lethally irradiated WT mice (C57BL/6 J mice). Four weeks after BMT, the recipient mice transplanted with the BM cells from the CalrWT/WT mice (WT-R) or Calrdel10/WT mice (del-R) were subjected to normoxia (21% O2) or chronic hypoxia (10% O2) for 3 weeks. d Allele frequency of the mutant Calr in the peripheral leukocytes of recipient mice at 4 weeks after BMT (n = 15, each). e Right ventricular (RV) systolic pressure (RVSP) and RV hypertrophy determined by dividing the RV weight by the left ventricular weight including the septum (RV/LV + S) (n = 6–8). f Representative hematoxylin–eosin (HE) staining and immunohistochemistry with antibodies to anti-α smooth muscle actin (αSMA) and anti-F4/80 images in the lung of BMT recipient mice from CalrWT/WT or Calrdel10/WT mice. Scale bars, 50 µm. g Quantitative analysis of the percentage of muscularized distal pulmonary arteries in αSMA-immunostained sections (n = 3, each). h Quantitative analysis of the pulmonary perivascular macrophages determined as F4/80-positive cells, per 30 vessels (n = 5, each). Data are presented as means ± SEM. d, e, g, h *P < 0.05 versus the corresponding normoxia group and P < 0.05 versus the corresponding BMT recipient mice from CalrWT/WT mice. WT-R, recipient mice transplanted with BM cells from CalrWT/WT mice; del-R, recipient mice transplanted with BM cells from Calrdel10/WT mice. Oligonucleotides and antibodies used are listed in Additional files 8, 9

In a public database [8], 138 CALR frameshifts, including the major del52 and ins5 [1], and another frameshift in 2 MPN patients (ET, myelofibrosis) exactly matching the Calr-del10, have been noted in patients with hematopoietic cancers, mostly MPNs (Additional file 7: Table S1, Additional file 1: Fig. S1f). Mouse models carrying frameshifted CALR showed ET or, rarely, myelofibrosis [9]. Likewise, Calrdel10/WT mice developed ET with phosphorylated STAT3 (pSTAT3) and cell-surface thrombopoietin-receptor (TpoR) expressions suggested to accompany mutant CALR [10], whereas Calrins2/WT mice showed a slight splenic enlargement (Fig. 1b, Additional files 2, 3: Fig. S2-S3).

To elucidate the roles of hematopoietic Calr mutation in PH, we performed non-competitive BMT from Calrdel10/WT mice (Fig. 1c), as we reconstituted Jak2V617F+ MPNs [11]. At 4 weeks after BMT, the engraftments were achieved in the BMT recipients from Calrdel10/WT mice (del-R, Fig. 1d), but their PB cell counts (Additional files 4: Fig. S4) and BM megakaryocytic distribution did not differ from BMT recipients from CalrWT/WT mice (WT-R). We assessed right heart hemodynamics and right ventricular (RV) hypertrophy, showing that neither RV systolic pressure (RVSP) nor right ventricle/left ventricle-plus-septum weight ratio (RV/LV + S) differed between WT-R and del-R. Subsequently, del-R were exposed to chronic hypoxia (10% O2) for 3 weeks. Strikingly, although chronic hypoxia elevated RVSP and RV/LV + S in both WT-R and del-R, these levels in del-R were significantly greater than in WT-R, suggesting that hematopoietic Calr mutation promotes PH (Fig. 1c-e).

Lung histology showed significant increases in PA medial wall thickness and muscularization, indicated by α smooth muscle actin, without thrombosis in del-R compared to WT-R under chronic hypoxia, whereas F4/80+ macrophages rather than TpoR+ cells were increased specifically in PA regions in both WT-R and del-R (Fig. 1f-h, Additional file 3: Fig. S3e). However, pSTAT3 levels were elevated in the lungs of del-R compared to WT-R after chronic hypoxia. The expression of Endothelin-1, an important vasoactive peptide involving PA remodeling in PH [4, 5], was also increased in the lungs of del-R compared to WT-R under chronic hypoxia (Fig. 2a, b, Additional file 5: Fig. S5). We visualized the Calrdel10/WT BM-derived cells using CAG-EGFP: in the lungs of BMT recipients from Calrdel10/WT/CAG-EGFP mice, donor-derived macrophages accumulated in PA regions, but donor-derived cells were not observed in vascular walls (Fig. 2c), suggesting that Calrdel10/WT BM-derived macrophages migrated into the PA regions.

Fig. 2
figure2

STAT3 phosphorylation and Endothelin-1 expression in the lung and macrophages from Calrdel10/WT mice. a Western blot of lung homogenates of the BMT recipients from CalrWT/WT mice (WT-R) or Calrdel10/WT mice (del-R), immunoblotted with the indicated antibodies. b Phosphorylated STAT3 (p-STAT3) to total STAT3 (t-STAT3) or Endothelin-1 to β-actin ratios are shown in the graphs. The average value for WT-R under normoxia was set to 1 (n = 5, each). *P < 0.05 versus the corresponding normoxia group and P < 0.05 versus the corresponding WT-R. c The lethally irradiated WT C57BL/6 J mice were transplanted with the BM cells from Calrdel10/WT/CAG-EGFP mice. These recipient mice were subjected to chronic hypoxia for 3 weeks, and then the lungs were fixed and stained with the indicated antibodies. Upper images show representative immunofluorescence of the lung sections stained with anti-GFP (green) and anti-αSMA (red) antibodies and DAPI (blue). Scale bars, 50 µm. Lower images show representative immunofluorescence of the lung sections stained with anti-GFP (green) and anti-F4/80 (red) antibodies and DAPI (blue). Scale bars, 10 µm. d-g BM mononuclear cells isolated from the CalrWT/WT or Calrdel10/WT mice were cultured in the presence of 10 ng/mL of M-CSF for 6 days. d Representative immunofluorescence images of the cells stained with anti-F4/80 (green) and DAPI (blue) are shown. More than 90% of cells were macrophages expressing F4/80. Scale bars, 25 µm. e Dot plot of flow cytometry for cultured macrophages. Red, blue, and orange dots represent cells from CalrWT/WT mice, Calrdel10/WT mice, and negative control (mixture of WT and Calr del10 cells), respectively. Over 90% WT and del10 cells were positive for both F4/80 and CD68. SSC indicates side scatter. f The cultured macrophages were then stimulated with 0.05 µg/mL of lipopolysaccharide (LPS), a potent activator of macrophages. The mRNA expression levels of Endothelin-1 (Edn1) were analyzed at the indicated time (n = 8, each). Actb was used for normalization. The average value for the macrophages from CalrWT/WT mice at baseline was set to 1. g Left panels show western blots on STAT3, Endothelin-1, and β-actin in the macrophages stimulated with 0.05 µg/mL of LPS. Right graphs show phosphorylated STAT3 (p-STAT3) to total STAT3 (t-STAT3) or Endothelin-1 to β-actin ratios at the indicated time. The average value for the macrophages from CalrWT/WT mice at the baseline was set to 1 (n = 4, each). All data are presented as means ± SEM. *P < 0.05 versus the corresponding WT group. WT, macrophages derived from the CalrWT/WT mice; del10, macrophages from the Calrdel10/WT mice. Oligonucleotides and antibodies used are listed in Additional files 8, 9

RNA sequencing of hematopoietic progenitors showed Calr-del10 activated JAK-STAT pathway, as well as cardiac-hypertrophy pathway that includes upregulation of Endothelin-1. Also, human CALR-del52 introduction upregulated Endothelin-1 in a macrophage cell line (Additional file 6: Fig. S6). We next obtained Calrdel10/WT macrophages by culturing BM-mononuclear cells (BM-MNCs) in the presence of M-CSF (Fig. 2d, e). The increases in the Endothelin-1 and pSTAT3 levels did not show the statistical difference between in CalrWT/WT and Calrdel10/WT macrophages at baseline, but these levels in Calrdel10/WT macrophages were significantly more upregulated compared to CalrWT/WT macrophages after lipopolysaccharide stimulation (Fig. 2f, g). These data suggest that BM-derived macrophages with the Calr mutation played important roles in the PA remodeling.

To date, most of studies for PH in MPN patients lack information about driver mutations, although a retrospective study indicated higher prevalence of CALR mutations in ET patients with PH than those without [6]. Besides megakaryocyte lineage with TpoR expression, a recent study indicated that transcriptional misregulation occurs with JAK-STAT activation in CALR-mutated PB-MNCs similar to JAK2-mutated PB-MNCs [12]. Our murine model revealed a hematopoietic phenotype with relevance to human MPNs with CALR mutations in terms of molecular mechanisms and PH. Further study of associations between CALR mutations and PH or macrophage activation is needed (Additional file 10).

Availability of data and materials

The RNA sequencing data have been deposited in the Gene Expression Omnibus database (GSE152482).

Abbreviations

CALR:

Calreticulin

ET:

Essential thrombocythemia

MPN:

Myeloproliferative neoplasm

PB:

Peripheral blood

BM:

Bone marrow

BMT:

Bone marrow transplantation

PA:

Pulmonary arterial

PH:

Pulmonary hypertension

Del-R:

BMT recipients from Calrdel10/WT mice

MNCs:

Mononuclear cells

RV:

Right ventricular

RVSP:

Right ventricular systolic pressure

RV/LV + S:

Right ventricle/left ventricle-plus-septum weight ratio

M-CSF:

Macrophage colony-stimulating factor

WT:

Wild type

WT-R:

BMT recipients from CalrWT/WT mice

References

  1. 1.

    Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369:2379–90.

    CAS  Article  Google Scholar 

  2. 2.

    Rabinovitch M, Guignabert C, Humbert M, Nicolls MR. Inflammation and immunity in the pathogenesis of pulmonary arterial hypertension. Circ Res. 2014;115:165–75.

    CAS  Article  Google Scholar 

  3. 3.

    Asosingh K, Farha S, Lichtin A, Graham B, George D, Aldred M, et al. Pulmonary vascular disease in mice xenografted with human BM progenitors from patients with pulmonary arterial hypertension. Blood. 2012;120:1218–27.

    CAS  Article  Google Scholar 

  4. 4.

    Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2013;62:D34-41.

    Article  Google Scholar 

  5. 5.

    Adir Y, Elia D, Harari S. Pulmonary hypertension in patients with chronic myeloproliferative disorders. Eur Respir Rev. 2015;24:400–10.

    Article  Google Scholar 

  6. 6.

    Lee M-W, Ryu H, Choi Y-S, Song I-C, Lee H-J, Yun H-J, et al. Pulmonary hypertension in patients with Philadelphia-negative myeloproliferative neoplasms: a single-center retrospective analysis of 225 patients. Blood Res. 2020;55:77–84.

    Article  Google Scholar 

  7. 7.

    Gomez-Arroyo J, Saleem SJ, Mizuno S, Syed AA, Bogaard HJ, Abbate A, et al. A brief overview of mouse models of pulmonary arterial hypertension: problems and prospects. Am J Physiol Cell Mol Physiol. 2012;302:L977–91.

    CAS  Article  Google Scholar 

  8. 8.

    Tate JG, Bamford S, Jubb HC, Sondka Z, Beare DM, Bindal N, et al. COSMIC: the catalogue of somatic mutations in cancer. Nucleic Acids Res. 2019;47:D941–7.

    CAS  Article  Google Scholar 

  9. 9.

    Shide K. The role of driver mutations in myeloproliferative neoplasms: insights from mouse models. Int J Hematol. 2020;111:206–16.

    CAS  Article  Google Scholar 

  10. 10.

    Masubuchi N, Araki M, Yang Y, Hayashi E, Imai M, Edahiro Y, et al. Mutant calreticulin interacts with MPL in the secretion pathway for activation on the cell surface. Leukemia. 2020;34:499–509.

    CAS  Article  Google Scholar 

  11. 11.

    Ueda K, Ikeda K, Ikezoe T, Harada-Shirado K, Ogawa K, Hashimoto Y, et al. Hmga2 collaborates with JAK2V617F in the development of myeloproliferative neoplasms. Blood Adv. 2017;1:1001–15.

    CAS  Article  Google Scholar 

  12. 12.

    Alimam S, Villiers W, Dillon R, Simpson M, Runglall M, Smith A, et al. Patients with triple-negative, JAK2 V617F- and CALR -mutated essential thrombocythemia share a unique gene expression signature. Blood Adv. 2021;5:1059–68.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Ms. Tomiko Miura and Ms. Shoko Sato, Department of Cardiovascular Medicine, Fukushima Medical University, Fukushima, Japan, Ms. Chisato Kubo and Ms. Ayumi Haneda, Office for Gender Equality Support, Fukushima Medical University, Fukushima, Japan, and Mr. Hiroshi Nakano, Center for Molecular Genetics, Yamagata University, Yamagata, Japan, for their technical assistance.

Funding

This work was supported by grants from JSPS KAKENHI to K.I. (15K09484, 18K08365), K.O. (15K09483) and T.Y. (19K17532), and the Uehara Memorial Foundation (201890006), and the Japanese Society of Hematology to K.I.

Author information

Affiliations

Authors

Contributions

K. Minakawa, K.U., T.Y., Y.K., T.M., K.W., Y.T., S.M., and Y.S. performed the research; O.N. generated mice; K. Mimura, K.O., T.I., and Y.T. analyzed and interpreted the data; Y.H. performed histological studies; K. Minakawa and K.E.N. interpreted the data and wrote the manuscript; K.E.N. provided editorial guidance; K.I. conceived the research, guided study design, analyzed and interpreted data, wrote the manuscript, and supervised study. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Kazuhiko Ikeda.

Ethics declarations

Ethics approval and consent to participate

The investigations conformed to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication, 8th Edition, 2011). All efforts were addressed to minimize suffering. All studies were approved by the Animal Study Committees of Fukushima Medical University (App. No. 245, 30086) and Yamagata University (App. No. 27-153, 28-113).

Consent for publication

Not applicable.

Competing interests

T.Y.’s and K.S.’s department receives support from Janssen Pharmaceutical K.K., Japan. T.M.’s department receives support from Fukuda Denshi Co., Ltd., Japan. These companies were not associated with the contents of this study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1. Fig. S1:

CALR proteins coded by ins2 and del10 frameshifts in murine Calr mimicked a feature of those coded by human type 2-like CALR mutations that generated novel C termini. a Western blot of BM cells using antibody specific for the CALR N terminus (CALR-N) or C terminus (CALR-C). b Isoelectric point (pI) in human and murine CALR proteins. c Alignment of C domains in mutant murine CALR from codon A352. Acidic and basic residues are in blue and red, respectively. #: the negatively charged amino acid stretches. : the subjects of previously reported murine CALR mutants. d-f Identity and similarity between murine and human CALR frameshifts. The 2 MPN patients, with a mutated protein as CALR p.K375Rfs*52 (c.1124_1133del), matched the murine Calr del10 (p.K375Rfs*52 coded by Calr c.1124_1133del), although identity and similarity of the peptides were slightly different (f).

Additional file 2. Fig. S2:

MPN-like phenotypes in knock-in mice with Calr frameshifts. a-b Body (a) and spleen (b) weights (n = 15—19). c BM nuclear cell counts (n = 4–6). d The proportions of BM CD71+Ter119+ erythroblasts, Gr1+ myeloid cells, B220+ B cells, and TCR+ T cells in flow cytometry (n = 3 in each). e–f Histology of BM (e) and spleens (f). g-h The numbers of megakaryocytes per high-power field (HPF) in BM (n = 3 in each) and spleens (n = 4—10). (*P < 0.05, **P < 0.01).

Additional file 3. Fig. S3:

Phosphorylation of STAT3 and expression of MPL, thrombopoietin receptor (TpoR). a Western blot of whole BM nuclear cells suspended in the absence of exogenous cytokines. b-c Flow cytometry gated with a lineage fraction in BM cells. b Overall expression of cell-surface TpoR. Left: Histogram; right: mean fluorescence intensity (MFI). c Cell-surface expressions of TpoR and CALR. Left: heatmap plots; right: proportions of cell-surface TpoR+ cells in association with CALR expression (n = 3 in each experiment; *P < 0.05; ns: no significant difference). d Immunofluorescence for MPL in bone marrow. e Immunofluorescence for MPL in lung. d-e Scale bars, 50 µm.

Additional file 4. Fig. S4:

Peripheral blood cell counts in the BMT recipients exposed to normoxia or chronic hypoxia for 3 weeks (n = 4–6). (*P < 0.05 versus the corresponding normoxia group).

Additional file 5. Fig. S5:

Relative Edn1 mRNA expression levels in the lung (n = 5, each). The average value for WT-R mice under normoxia was set to 1. (*P < 0.05 versus the corresponding normoxia group, and P versus the corresponding WT-R mice under chronic hypoxia)

Additional file 6. Fig. S6:

Gene expressions. a-e RNA sequencing (RNAseq) in LSK (lineageSca1+c-Kit+) cells of an aliquot from 4 male mice of 3 months age in each sample from Calrins2/WT mice, Calrdel10/WT mice, and CalrWT/WT mice. a Principle component analysis. b Venn diagrams of upregulated and downregulated genes (> twofold) in LSK cells of Calrdel10/WT mice or Calrins2/WT mice relative to those of CalrWT/WT mice. c Pathway analysis by the Ingenuity Pathway Analysis software (Qiagen). All the pathways in the comparison analysis of canonical pathways with both Z score ≥|2| and p < 0.05 in at least one of the Calrins2/WT mice and Calrdel10/WT mice relative to CalrWT/WT mice are shown. • indicates the box which did not reach the level of Z score ≥|2| in the genotype shown. The allow indicates Cardiac Hypertrophy Signaling pathway upregulated in both Calrins2/WT mice and Calrdel10/WT mice. d Individual genes in the Cardiac Hypertrophy Signaling pathway. Differentially expressed genes ( >|10|-fold) in Calrdel10/WT mice relative to CalrWT/WT mice, including EDN1 that codes Endothelin-1 (allow), are shown. e Gene set enrichment analysis (GSEA) for the JAK-STAT pathway. NES indicates normalized enrichment score; FDRq, false discovery rate q value. f-g Introduction of FLAG-Tag-inserted human WT and del52 CALR constructs into a macrophage cell line, RAW 264.7. f Western blots. g The levels of Endothelin-1 mRNA (Edn1) were analyzed in RAW 264.7 cells introduced with CALR WT or del52 after incubation under normoxia (21% O2) or hypoxia (10% O2) for 24 h. Samples were taken from 3 wells for each experiment. Actb was used for normalization. The average value for cells introduced with WT CALR and incubated under normoxia was set to 1.

Additional file 7. Table S1:

Frameshifts in CALR exon 9 on the COSMIC database in hematopoietic cancers.

Additional file 8. Table S2:

Oligonucleotides used in this study.

Additional file 9. Table S3:

Antibodies used in this study.

Additional file 10:

Supplementary methods, results, and references.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Minakawa, K., Yokokawa, T., Ueda, K. et al. Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice. J Hematol Oncol 14, 52 (2021). https://doi.org/10.1186/s13045-021-01064-8

Download citation

Keywords

  • CALR
  • Pulmonary hypertension
  • Myeloproliferative neoplasms
  • Macrophage
  • Essential thrombocythemia
\