Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms

Abstract

Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.

Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN

“…Role of homozygosity in the expansion of CALR ins5 HSCs. On one hand, platelet counts were reported to be higher in ins5-ET than in del52-ET patients 16,17 and, on the other hand, homozygous clones are more frequently found in ins5than in del52mutated patients 2,3,18,19 . Thus, we decided to investigate how homozygous ins5 cells might out-compete heterozygous ins5 cells at the HSC level and how it would affect platelet levels knowing that thrombocytosis in homozygous ins5/ins5 mice was significantly superior than in heterozygous +/del52 mice (Fig.…”

“…Although allele burden in CALR-mutated patients often tends to be close to 50% when disease declare, it would be interesting to compare platelet levels in del52 and ins5 ETs with similar and low allele burden and to follow progression of thrombocytosis to measure the real impact of these two mutations on platelet counts. Third, it was reported that homozygous CALR mutations, although they are rare, were rather ins5 and type 2-like than del52 and type 1-like mutations 2,3,18,19 . In ET patients, homozygous ins5 is frequently restricted to a small fraction of progenitors, but may contribute to high level of thrombocytosis.…”

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms

“…Here, we provide evidence that CALRdel52 and CALRins5 iPSC reproduce the MK hyperplasia observed in patients and in mouse models. 7 – 9 , 15 , 22 , 37 We observed an increase in the generation of MK, a TPO hypersensitivity of the CFU-MK progenitors as well as an increase in their size in CALR -mutated iPSC derived from 4 different patients compared with control iPSC, suggesting a stronger MK precursor proliferation and/or a generation of more immature MK progenitors. Thus, CALR mutations modify the MK differentiation at multiple steps.…”

“… 20 They frequently occur after CALR mutations in hematopoietic stem cells. 21 , 22 They are “modifying mutations” that change the phenotype or accelerate the disease. 23 …”

CALR frameshift mutations in MPN patient-derived iPS cells accelerate maturation of megakaryocytes

“…We confirmed the upregulation of typical megakaryocytic genes such as NFE2, FLI1 and VWF in our iPS cell-derived MKs by RT-qPCR analysis and proceeded to an in-depth analysis of the global gene expression profile by RNAseq. Recent studies reported on MPN-related transcriptome analysis of in vitro differentiated MKs from primary CALR -mutated CD34+ (El-Khoury et al, 2020) or single-cell RNAseq of primary CD34+ cells from MF patients with either JAK2 V617F or CALR mutation (Psaila et al, 2020). In contrast to the first mentioned study, we were able to relate differences of the megakaryocytic transcriptome to the underlying CALR genotype by analyzing a clonal cell population.…”