Amplification of Von Willebrand Factor-Positive <i>CALRmut</i> Disease-Initiating Hematopoietic Stem Cells Is Associated to the Activation of the PERK/EiF2a Branch of Unfolded Protein Response

Introduction The 2 most frequent somatic mutations in the calreticulin (CALR) gene associated to essential thrombocythemia and myelofibrosis patients are a 52 base-pair (bp) deletion (del52) and a 5 bp insertion (ins5). We previously modeled both types of CALR mutations (CALRmut) in knock-in (KI) mice and observed a more severe megakaryocytic (MK) phenotype with CALRdel52 than with CALRins5, including a strong amplification and competitive advantage of CALRdel52 hematopoietic stem cells (HSC). Both types of CALRmut acquire a new C-terminal tail that stabilizes their interaction to the thrombopoietin receptor, stimulating its oncogenic signaling at the cell surface. Moreover, CALRmut lose their endoplasmic reticulum (ER) retrieval KDEL motif leading to their secretion, ER stress and activation of the unfolded protein response (UPR). However, the disease-initiating cells within the HSC compartment have not been identified and the precise mechanism of action of CALRmut remains incompletely understood. These results are essential to enable effective therapeutic targeting. Methods To analyze the effects of CALRmut in HSC, we used a combination of single cell techniques (scRNAseq and mass cytometry) and KI mice expressing the murine CALRdel52 or CALRins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase. These KI mice were crossed with transgenic mice expressing the GFP under the control of the von Willebrand factor (Vwf) promoter. Results We combined scRNAseq analyses of mouse LSK progenitors and results from a trajectory inference algorithm on mass cytometry data. We observed on a UMAP plot an expansion of HSC (1.8-fold) and multipotent progenitors MPP2-3 (1.5-fold) with CALRdel52 and MPP1 (1.2-fold) with CALRins5 at the expense of lymphoid-primed MPP4 (4.4-fold and 1.8-fold less with CALRdel52 and CALRins5, respectively). Consistent with the stronger amplification of CALRdel52 HSC, we found a significant upregulation of Ki-67 in these cells and a higher proliferation index compared to wild-type (WT) and CALRins5 HSC (4.72 vs 3.22 and 3.45). We also detected a significant activation of eiF2a from the PERK branch of the UPR in CALRdel52 but not in CALRins5 HSC, which decreased to WT level upon MK differentiation. Interestingly, we observed an upregulation of vWF gene expression in CALRdel52 HSC, indicating a platelet/myeloid bias. Differential analysis between vWF-positive (vWF+) HSC, that are at the top of hematopoiesis, and vWF-negative (vWF-) HSC pointed to the upregulation of a UPR signature in the vWF+ context that was exacerbated by CALRdel52. Using capillary electrophoresis and qPCR, we confirmed in CALRdel52 KI mice carrying the Vwf promoter-GFP construct that eiF2a was predominantly phosphorylated and activated in vWF+vs vWF- HSC. The function of these sorted vWF- or vWF+ HSC was analyzed after engraftment in lethally-irradiated recipient mice. Both groups of mice developed thrombocytosis, but mice engrafted with vWF+CALRmut HSC had a more pronounced disease phenotype, particularly with CALRdel52 (n=7-10; 4,893±299 vs 3,283±398 x103/mL platelets at 20 weeks after CALRdel52 expression in vWF+vs vWF- HSC and 2,180±112 vs 1,462±104 for CALRins5). Secondary and tertiary bone marrow transplantations (n=5-10) undoubtedly identified vWF+CALRmut HSC rather than vWF- HSC as the disease-initiating cells. The vWF+ HSC can directly differentiate into MK via a biased route or in a stepwise manner via the generation of vWF- HSC. CALRmut, especially CALRdel52, led to a significant expansion of vWF+ HSC without exacerbating their platelet bias. Notably, both CALRmut conferred platelet-biased properties to the vWF- HSC while retaining their lymphoid potential. Conclusions In conclusion, using CALRmut mouse models, we demonstrate that thrombocytosis is initiated from the amplification of the disease-initiating platelet-biased vWF+ HSC and the increased capacity of vWF- HSC to give platelets. The differential amplification of CALRdel52vsCALRins5 HSC might in part be explained by the activation of the PERK/eiF2a branch of the UPR in the vWF+CALRdel52 HSC sub-population. These results highlight a new cell of origin and a mechanism of action of CALRdel52 in HSC that could be targeted as part of a therapeutic approach.