Calreticulin Mutants Induce an Early Clonal Dominance and a Megakaryocytic Phenotype through the Activation of MPL/JAK2 Pathway in Human Primary Cells

Myeloproliferative neoplasms (MPNs) are clonal malignant disorders characterized by the increased production of mature myeloid cells in blood. The classical MPNs include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Those pathologies are due to the acquisition of gain-of-function mutations leading to the constitutive activation of the cytokine receptor / JAK2 signaling pathway: JAK2V617F in 70% of cases and mutations in the thrombopoietin receptor (MPL) gene in 5% of cases. More recently, around fifty different mutations in the calreticulin (CALR) gene have been described in 30% of ET and PMF with two more frequent mutations called del52 (type 1) and ins5 (type 2). All the CALR mutations induce a frameshiflt to an alternative reading frame in the exon 9 leading to a new C-term tail of the protein with hydrophobic features, and the loss of the KDEL sequence, which is involved in its endoplasmic reticulum retention.

The goal of this work was to understand the role of CALR mutants (del52, del46, del34, ins5, del19, del13) in human hematopoiesis.

By studying the variant allele frequency (VAF) in 20 patients, we have shown that the CALR mutations are present in all blood mature cells not only in granulocytes and monocytes (CD14⁺) with a VAF >30% but also in B cells (CD19⁺), NK cells (CD56⁺) and in some cases in T cells (CD3⁺). Moreover, we have observed that CALR mutations are present in all hematopoietic progenitors including CD34⁺CD38^-CD90⁺ (HSC), CD34⁺CD38^-CD90^- (immature progenitors) and CD34⁺CD38⁺ (committed progenitors) cell fractions after investigating the clonal architecture of the progenitors. CALR mutation was detectable in more than 40% of progenitor cells except in 2 patients (15 patients studied) and with, in some cases, no detectable wild type CALR progenitors. Homozygous CALR mutations were rare except in one case associated with disease progression. Whatever the VAF, there was no significant differences among the different progenitor types and granulocytes. Finally, we observed that all the associated mutations studied (TET2, PHF6, SYNE1, SCARA5, PIK3CD, SETD1B) in 6 patients postdated CALR mutations. We could also show in 15 patients samples that CALR mutants give a specific megakaryocytic progenitor (CFU-MK) spontaneous growth mediated both by MPL and JAK2 activation using specific inhibitors and short hairpin RNAs. The CFU-MK spontaneous growth correlated with a constitutive activation of JAK2/STAT3/5 pathway in megakaryocytes derived from in vitro cultures of CD34⁺ progenitors.

In aggregate, these results show that all CALR mutants studied are present in all human hematopoietic cells including myeloid and lymphoid cells, give an early clonal advantage at the level of the HSC compartment and a specific increased growth of the megakaryocytic lineage via MPL/JAK2 activation.