Haploinsufficiency of Calreticulin Causes Dysregulation of Hematopoiesis and Results in a Myeloproliferative Phenotype in an in-Vivo Mouse Model

Abstract

Background.JAK2V617F, MPLW515K/L and CALR exon9 mutations drive the development of Philadelphia negative Myeloproliferative Neoplasms (MPN). While JAK2 and MPL knock out (KO) leads to severe defects of hematopoiesis, the role of Calreticulin (CALR) deregulation is not fully understood, however recent reports demonstrated that CALR deletion leads to neoplastic transformation (Shide K et al Blood, 2020; Balliu M et al Blood Adv., 2021). Aim. To investigate the role of CALR in hematopoiesis and MPN development, we generated and characterized a Calr KO mouse model. Methods. We developed a Calr+/- mouse model by crossing mice carrying a floxed allele targeting Calr intron 3 and 7 (Calr+/flx) with Vav-Cre mice. The phenotypic characterization was performed by blood counts analysis, bone marrow (BM) and spleen histology, flow-cytometry and clonogenic assay by plating 2x104 cells for CFU-GEMM, CFU-GM and BFU-E and 2.5x105 for CFU-Mk. Proteome profiler membrane-based array and RNA-sequencing were employed to profiling BM protein and gene expression. Results. Sanger sequencing of BM cells of Calr+/- mice confirmed the deletion of Calr exons 4-7, resulting in the downregulation of Calr mRNA (-0.56-fold ±0.1) and decreased protein content (-0.47-fold ±0.21) compared to Calr+/flx mice (hence, controls). The productivity, body weight and lifespan were comparable between Calr+/- and controls. Calr+/- mice showed increased platelet count (median: 1360, range: 1156-1986 x109/L), 1.2 to 1.5-fold compared to controls, starting from month 4 (p=0.007) up to the latest time point available at 12 months (p=0.03). Erythrocytes count of Calr+/- increased after month 6 up to month 12 (median:10.3, range:9.9-10.6 x1012/L)(P=0.004) paralleled by an upward trend of hematocrit (median:47%, range:42-51%) and hemoglobin (median:18.3, range:14.2-20.1 g/L) levels. The spleen index (spleen:body weight ratio x100) resulted increased in Calr+/- (mean: 0.38±0.09) compared to controls (mean: 0.3±0.08) (P=0.039). Histology revealed an augmented number of poly-lobulated megakaryocytes, most of which found in clusters, in BM of Calr+/- mice. Most Calr+/- mice showed a progressive loss of spleen architecture with resulting loss of clear demarcation between red and white pulp combined with focal fibrosis, especially in aged mice. Calr haploinsufficiency conferred an augmented clonogenic capacity to BM and, to a lesser extent, splenic progenitors, in semisolid culture assay. BM Calr+/- CFU-GEMM, CFU-GM and CFU-Mk were in median 8.9 (5-15), 102.7 (49-157) and 16 (9.7-13.6) and 5.7 (3 -10) (P=0.007), 80.2 (33-159) (P=0.0031) and 11.9 (12.7-22) (P=0.048) in Calr+/flx, respectively. Of note, BM BFU-E count was 1.3-fold higher in Calr +/- than controls (P=0.009). Calr+/- splenic progenitors showed an enhanced clonogenic potential, strikingly for CFU-GEMM (P=0.0064). We observed that Calr+/- myeloid progenitors were significantly expanded when compared to controls: BM Mk precursors (FcγRlow/cKIT+/CD41+) and both BM and spleen LSK (Lin-/Sca1+/cKIT+) were, respectively, +1.6-fold (P=0.048), +1.4-fold (P=0.042) and +2-fold (P=0.008) than controls. Proteomic array performed on BM cells identified a set of deregulated proteins, mainly represented by cytokines (IL1α, IL4, IL6), chemokines (CXCL5, CXCL12, C1qR, VEGF, Endoglin) and cell adhesion molecules (VCAM, ICAM). These observations were strengthened by the result of transcriptomics analysis on bulk RNA-sequencing from Calr+/- and Calr+/flx mice BM cells highlighting a number of differentially expressed RNAs (increased n=385, decreased n=350) that resulted enriched in signaling pathways regulating chemokines and cell adhesion molecules, including "cell chemotaxis” (GO:0060326), "leukocyte migration” (GO:0050900), "regulation of cell-cell adhesion” (GO:0022407); cytokines, as determined by "Cytokine-cytokine receptor interaction” (KEGG:mmu04060); and Ca2+ homeostasis, including "cellular calcium ion homeostasis” (GO:0006874), "regulation of cytosolic calcium ion concentration” (GO:0051480 ). Conclusion. Finding from this in-vivo model suggest that haploinsufficiency of Calr induces dysregulation of hematopoiesis, ultimately resulting in a mild-ET phenotype. These findings will be validated in larger series and integrated with ongoing experiments to outline the molecular mechanisms underlying the CALR-driven MPN phenotype.