Paper Details

ABSTRACT

Background: Calreticulin (CALR) is a major endoplasmic reticulum (ER) chaperone and calcium (Ca2+)-buffering protein that plays a central role in proteostasis and intracellular Ca2+ homeostasis. Somatic CALR mutations are established drivers of myeloproliferative neoplasms (MPN); however, consequences of quantitative CALR reduction remain incompletely understood.

Aim: To investigate whether CALR haploinsufficiency per se is sufficient to perturb wild-type hematopoiesis and/or contribute to an MPN-like phenotype.

Methods: We generated and characterized a conditional Calr floxed (flx) mouse model on a C57BL/6 background and achieved hematopoietic-specific deletion (Calr+/-) through Vav1-iCre–mediated excision.

Results: Heterozygous Calr+/− mice displayed ≈50% reduction of Calr mRNA and protein level in bone marrow (BM) cells. Aging Calr+/− mice developed an MPN phenotype characterized by sustained thrombocytosis without changes in other blood cell lineages. Platelet (Plt) counts started to be significantly elevated since month +2 remaining persistently elevated up to last observation point (month +15; mean 1.3±0.07-fold vs Calr+/flx, P<0.0001). BM histopathology revealed marked megakaryocyte (Mk) hyperplasia with clustered Mk associated with grade II–III reticulin fibrosis, while other lineages were normal. Calr+/− mice exhibited mild splenomegaly, peaking between month +8 (1.4-fold vs flx, P=0.02) to +10 (1.8-fold, P=0.004); histology showed extramedullary hematopoiesis with fibrotic remodeling. Increased numbers of CFU-GEMM, CFU-GM and CFU-Mk (1.5-fold, P<0.0001; 1.3-fold, P=0.0029; 1.7-fold, P<0.0001, vs flx respectively) were generated by BM progenitors whereas erythroid output was preserved (0.99-fold, P=0.8). Expansion of the pool of BM and spleen Lin−Sca-1+c-Kit+ stem/progenitor cells (+1.3-fold and +2-fold, respectively; P<0.05) and BM Mk progenitors (FC?Rlowc-Kit+CD41+, +2.3-fold, P=0.04) was documented. Unsupervised analysis of RNA profile by RNAseq of Lin− cells revealed a coherent deregulated gene set enriched for cell adhesion, Ca2+ homeostasis, and MAPK/PI3K signaling, consistent with transmission electron microscopy (TEM) ultrastructural findings of increased cell-to-cell interactions via membrane pseudopodia and adherens junctions in BM sections. Phospho-proteomics (LC–MS/MS) of Calr+/- BM myeloid cells identified 179 deregulated phosphosites (|LFC|>0.58, P<0.05) clustering into six groups, with AURKAb, CDK4, CDK6, and AMPK as central hubs; marked AMPK phosphorylation. Western blot analysis of BM cells confirmed increased MAPK/PI3K activation, with overall mild JAK/STAT changes and preferential STAT3 activation. Mks generated in vitro from Calr+/- Lin− cells showed spontaneous Ca2+ oscillations of higher frequency/amplitude vs flx, more cells with active Ca2+ flux, and deregulated influx/efflux These Mks also displayed exaggerated proplatelet extensions and abnormal Plt morphology. TEM of Calr+/- BM revealed Mk maturation defects (vacuolated cytoplasm without demarcation membrane; Plt field–like regions lacking typical granules). Newly formed Plts in the BM showed irregular shape, dense pseudopodia, reduced granule content, and immature features (prominent mitochondria, incomplete membrane thickening). Blood Plts were characterized by significantly higher surface P-selectin (CD62P) expression in unstimulated Calr+/- Plts, indicating constitutive activation. Plts RNAseq identified 422 DEGs (|LFC|>1, P<0.05) separating Calr+/- from flx phenotypes, with GSEA showing pervasive negative enrichment of lipid/sterol metabolism (i.e.: GO:0008202 NES:−2.19, FDR:0.007; GO:0055092 NES:−2.00, FDR:0.02), alongside suppressed complement/coagulation cascades (hsa04610 NES:−2.20, FDR:3.7×10?5; HALLMARK_COAGULATION NES:−1.85, FDR:0.02), and enrichment of the Ca2+/PKCα/NF-κB axis, linking ER Ca2+ dysregulation to impaired metabolic programming and defective Plts function.

Conclusions: Current observations in an original Calr+/- mouse model indicate that Calr haploinsufficiency alone is sufficient to induce an MPN-like disorder marked by thrombocytosis with Mks expansion and BM fibrosis, stem/progenitor activation, Ca2+ signaling instability, metabolic rewiring, and Plts dysfunction. Overall, findings suggest that quantitative loss of canonical Calr function, independent of neomorphic mutations, can per se initiate pathological myeloproliferation via disrupted ER-Ca2+ coupling and downstream kinase networks.