In this issue of Blood, Shide et al separate the roles of loss of a normal CALR
allele and gain of a mutant CALR allele in CALR-driven essential thrombocythemia (ET)

Green, Anthony R

Prins, Daniel

English

Apollo (Cambridge)

Mutant CALR functions: gains and losses  Daniel Prins and Anthony R. Green, Wellcome – MRC Cambridge Stem Cell Institute, University of Cambridge  In this issue of Blood, Shide et al. separate the roles of loss of a normal CALR allele and gain of a mutant CALR allele in CALR-driven essential thrombocythaemia (ET)1. Approximately one in four patients with ET, a blood cancer characterized by over-production of platelets, has a frameshift mutation in the gene encoding calreticulin (CALR)2,3. Research to date has demonstrated a gain-of-function role for the frameshifted CALR protein in binding to the thrombopoietin receptor (TpoR), thus acting as a rogue ligand and triggering proliferation and megakaryopoiesis. However, there has been less investigation of the effects of the loss of a normal CALR allele: CALR-/- mice are embryonic lethal due to malformation of the heart, preventing study of haematopoiesis in these mice4.  Shide et al. generated a mouse with haematopoietic-specific deletion of the CALR allele to allow separation of gain- and loss-of-function effects of the CALR mutation. Loss of one or both CALR alleles in haematopoietic cells had little effect on bone marrow haematopoiesis and did not cause ET, though loss of both alleles increased extramedullary haematopoiesis. In transplantation experiments, loss of one or both alleles increased bone marrow repopulation in primary recipients, but only loss of a single allele maintained this advantage in secondary transplants, indicating that CALR haploinsufficiency yields a competitive advantage. The authors further demonstrate that a CALRdel52 transgene can only drive a MPN phenotype (thrombocytosis) in transplants when WT CALR is haploinsufficient. Finally, they use transcriptomic data to investigate potential mechanisms underlying the advantage of CALR+/- cells. Compared to WT stem/progenitor cells, they find that CALR+/- cells have increased expression of E2F targets, suggestive of higher cell cycling. When comparing WT and CALR+/- cells also expressing transgenic CALRdel52, the authors again show upregulation of E2F target genes, in addition to an increase in stem cell self-renewal pathways and decreases in pathways responsive to the pro-inflammatory cytokines TNF-α and IFN-γ.  These results advance the field in several ways, not least by demonstrating a previously unknown role for normal CALR in haematopoiesis. Additionally, they shed light on important questions about differences between mouse models and human patients: while human patients typically show clonal disease, mouse models to date have shown either no5,6 or a slow-rising7 competitive advantage. The results of Shide et al. suggest that an approach wherein two WT CALR alleles are maintained6 may show no competitive advantage, as this requires loss of a normal CALR allele. Therefore, knock-in approaches may be more informative, as they more closely resemble patients. One mouse model with knock-in of a humanized CALRdel52 allele gave rise to a strong ET phenotype but no advantage within transplants5. A separate CRISPR-Cas9-based mouse model of the CALRdel52 mutation within the mouse CALR gene exhibited a lower platelet phenotype and a slow-rising competitive advantage in primary transplants, consistent with the requirement for CALR haploinsufficiency to yield a stem cell advantage7. The differences between the models remain unresolved, but may reflect differences in how murine and human frameshifted CALR protein bind to TpoR7 or other differences in the generation of the models. Furthermore, the results of Shide et al. emphasize that the level of CALRdel52 expression is crucial: progression to myelofibrosis in mouse models is only seen when CALRdel52 is highly expressed, as via retroviral expression6 or homozygosity of the knocked-in CALRdel52 allele5. Together, these results underscore the importance of ensuring that model systems resemble the situation in human patients, who have one mutant and one wild-type allele. Similarly, while results from transplant experiments can give insight into stem cell function, it is important to remember that these are artificial settings that do not resemble steady state haematopoiesis in patients. Going forward, more research will be necessary to understand why CALR haploinsufficiency is so critical to bestow a competitive advantage on stem cells: while there are indications of increased cell cycling and self-renewal, no direct mechanism has been proposed. It remains to be seen if this mechanism will rely on one of the canonical functions of CALR, such as protein chaperoning or calcium signalling8, or if the mechanism will be as surprising as the discovery of TpoR activation by frameshifted CALR protein. As the transcriptomic data were obtained from stem/progenitor cells from transplant recipients, they may be confounded by the stresses of the transplantation protocol on both donor cells and the recipient bone marrow niche. Overall, this study is an important advance in our understanding mutant CALR-driven ET: in addition to the well-studied role of mutant CALR as a rogue ligand for TpoR, Shide et al. show a role for the loss of a wild-type CALR allele that warrants further investigation.    References 1. Shide K. Calreticulin haploinsufficiency augments stem cell activity and is required for onset of myeloproliferative neoplasms. Blood. 2020;  2. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2. N. Engl. J. Med. 2013;369(25):2391–2405.  3. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic Mutations of Calreticulin in Myeloproliferative Neoplasms. N. Engl. J. Med. 2013;369(25):2379–2390.  4. Mesaeli N, Nakamura K, Zvaritch E, et al. Calreticulin is essential for cardiac development. J. Cell Biol. 1999;144(5):857–868.  5. Li J, Prins D, Park HJ, et al. Mutant calreticulin knockin mice develop thrombocytosis andmyelofibrosis without a stemcell self-renewal advantage. Blood. 2018;131(6):649–661.  6. Marty C, Pecquet C, Nivarthi H, et al. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis. Blood. 2016;127(10):1317–1324.  7. Balligand T, Achouri Y, Pecquet C, et al. Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development. Leukemia. 2020;34(2):510–521.  8. Michalak M, Groenendyk J, Szabo E, Gold LI, Opas M. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem. J. 2009;417(3):651–666.   

Mutant CALR functions: gains and losses.

https://www.repository.cam.ac.uk/bitstream/1810/308107/1/Commentary%20text%202.0.pdf

Mutant CALR functions: gains and losses.

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