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Calreticulin haploinsufficiency is needed for the induction of MPN-like pathology in vitro

Jun 19, 2020
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In more than 80% of patients with myeloproliferative neoplasms (MPN), mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus (MPL), and calreticulin (CALR) genes have been reported. These mutations seem to contribute to the pathogenesis of MPN through the constitutive activation of the JAK/signal transducer and activator of transcription (STAT) signaling pathway.1 Mutant CALR has been shown to bind to MPL and activate downstream signaling pathways that lead to the induction of MPN pathology in mouse models.2 As far as the role of these genes in normal hematopoiesis is concerned, knockdown of Jak2 or Mpl in mouse models completely abolishes hematopoiesis.2 However, the effect of CALR deficiency in normal hematopoiesis remains unknown, as Calr-knockout animals die before birth.

To determine the role of CALR in normal hematopoiesis, Kotaro Shide et al.2 generated a mouse model in which only hematopoietic cells lack Calr expression. The authors also further explored the role of CALR in MPN pathogenesis by generating CALRdel52 mutant mice with Calr haploinsufficiency mimicking human MPN. The results of this study were published in Blood and are summarized below.

Study design

Transgenic mouse models

  • Mx1-cre mice were crossed with Calr+/- and their offspring to Calrfl/- mice, which carried a floxed allele that targeted exons 4-7 of Calr, to generate Mx1-Cre;Calrfl/- (Calrfl/-) mice
  • In these mice, Calr was exclusively knocked out from Mx1 expressing cells (hematopoietic lineage cells) by a floxed allele deletion using intraperitoneal injection of polyinosinic–polycytidylic acid (400 μg) every other day in 4-week-old mice
  • Lethally irradiated B6-CD45.1 mice were used for bone marrow transplantation experiments
  • Mice were analyzed when 46 months old

Results

Exclusive CALR knockdown in hematopoietic cells induces extramedullary hematopoiesis

Bone marrow findings:

  • Generation of Calrfl/- mice, in which Calr is exclusively deleted from hematopoietic lineage cells revealed the following:
    • The femur and bone marrow cell pellet from Calrfl/- mice appeared anemic, with lower numbers of erythroid cells and colony-forming units in vitro
    • Calrfl/- mice had higher numbers of bone marrow multipotent progenitors and granulocytemacrophage progenitors
    • No changes in peripheral leukocytes, platelets, or hemoglobin levels
    • No change in numbers of myeloid lineage cells, B cells, or T cells
    • Comparable bone marrow cellularity and megakaryocyte numbers
    • Full megakaryocyte maturation was observed in both groups with no morphological differences
    • No differences in bone marrow mature myeloid cells (Mac1+, Gr1+), T cells (CD3+), B cells (B220+), or megakaryocytes (CD41+)

Spleen findings:

  • Contrasting the above-mentioned minor differences observed in the bone marrow of Calrfl/- mice, significant splenomegaly and extramedullary hematopoiesis was detected in these mice:
    • Calrfl/- mice displayed an almost twofold heavier spleen with no apparent border between the white and red pulp
    • There was heavy splenic infiltration of the red pulp by maturing myeloid cells and megakaryocytes
    • There was significant splenic increase in hematopoietic stem cells, erythroid cells (CD71+/Ter119+), and most myeloid progenitor cells
  • No anemia was observed in the Calrfl/- mice, which was hypothesized to be due to the compensatory splenic hematopoieisis seen in these mice
  • No leukemia onset was detected in the Calrfl/- mice, which did not have a different 2-year survival from their control counterparts

CALR haploinsufficiency increases the self-renewal of hematopoietic stem cells

  • To investigate the functional significance of CARL on hematopoietic stem cells, the authors performed a serial transplantation assay where bone marrows from either Mx1-cre;Calr+/-, Mx1-cre;Calr+/+, or Calrfl/- mice were transplanted into lethally irradiated recipient mice together with an equal amount of wild type bone marrows and then serially transplanted into further recipients
  • The results showed that there was a significantly higher self-renewal capacity in the hematopoietic stem cells derived from the Mx1-cre;Calr+/- mice, that was obvious in both the first and second recipient transplantations. This result indicates that CALR haploinsufficiency, but not loss of CALR, increases the capacity of hematopoietic stem cells for regeneration

CALR haploinsufficiency is needed for CALR mutation-induced MPN pathology

  • Since CALR haploinsufficiency was shown to increase the self-renewal of hematopoietic stem cells, the authors then sought to investigate its role in MPN mutated mice (CALRdel52)
    • Serial bone marrow transplantation assay showed that hematopoietic stem cells from CALRdel52;Calr+/+ mice had lower self-renewal capacity than controls
  • To examine whether CARL haploinsufficiency can restore this lower self-renewal capacity of CALRdel52 mutant mice, the authors generated CALRdel52;Calr+/- mice
    • These mice showed significant thrombocytosis and increased hematopoietic stem cell numbers in the bone marrow, indicating that CARL haploinsufficiency not only restores the hematopoietic damage induced by the CALRdel52 mutation but also induces an MPN-like phenotype
    • In support of the previous results, CALRdel52;Calr+/+ cells showed decreased repopulating capacity than controls when transplanted into healthy irradiated recipients, while CALRdel52;Calr+/- cells showed significantly higher expansion than control cells for up to three moths posttransplantation
  • This finding was further validated by gene expression profiling, showing that the stem cell gene signature with self-renewing genes and an inflammation signature with TNF-a and IFN-g signaling genes was significantly increased in CALRdel52;Calr+/- compared to CALRdel52;Calr+/+ cells
  • These results indicate that the CALRdel52 mutation contributes to MPN pathogenesis only in the presence of CALR haploinsufficiency

Conclusion

The results of this preclinical study indicate that lack of CALR expression exclusively in hematopoietic cells results in significant extramedullary hematopoiesis in the spleen. Moreover, the authors showed that CALR haploinsufficiency enhances the self-renewing capacity of hematopoietic stem cells and compensates for the reduced self-renewal capacity induced by the CALRdel52 mutation. Furthermore, only mice carrying both the CALRdel52 mutation and CALR haploinsufficiency (as seen in patients) develop an MPN-like phenotype with thrombocytosis, increased numbers of megakaryocytes, and enhanced growth of bone marrow stem cells.

  1. Jang M, Won Choi C. Recent insights regarding the molecular basis of myeloproliferative neoplasms. Korean J Intern Med. 2020;35(1):1-11. DOI: 10.3904/kjim.2019.317
  2. Shide K , Kameda T, Kamiunten A, et al. Calreticulin haploinsufficiency augments stem cell activity and is required for onset of myeloproliferative neoplasms. Blood. 2020;blood.2019003358 DOI: 10.1182/blood.2019003358

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