Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Inferring the initiation and development of myeloproliferative neoplasms.

The developmental history of blood cancer begins with mutation acquisition and the resulting malignant clone expansion. The two most prevalent driver mutations found in myeloproliferative neoplasms-JAK2V617F and CALRm-occur in hematopoietic stem cells, which are highly complex to observe in vivo. To circumvent this difficulty, we propose a method relying on mathematical modeling and statistical inference to determine disease initiation and dynamics. Our findings suggest that CALRm mutations tend to occur later in life than JAK2V617F. Our results confirm the higher proliferative advantage of the CALRm malignant clone compared to JAK2V617F. Furthermore, we illustrate how mathematical modeling and Bayesian inference can be used for setting up early screening strategies.

Inferring the dynamics of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.

In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target.

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients' erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV. SIGNIFICANCE: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265.

A multi-decadal record of oceanographic changes of the past ~165 years (1850-2015 AD) from Northwest of Iceland.

Extending oceanographic data beyond the instrumental period is highly needed to better characterize and understand multi-decadal to centennial natural ocean variability. Here, a stable isotope record at unprecedented temporal resolution (1 to 2 years) from a new marine core retrieved off western North Iceland is presented. We aim to better constrain the variability of subsurface, Atlantic-derived Subpolar Mode Water (SPMW), using near surface-dwelling planktic foraminifera and Arctic Intermediate Water (AIW) mass changes using benthic foraminifera over the last ~165 years. The reconstruction overlaps in time with instrumental observations and a direct comparison reveals that the δ18O record of Neogloboquadrina pachyderma is reliably representing temperature fluctuations in the SPMWs. Trends in the N. pachyderma δ13C record match the measured phosphate concentration in the upper 200 m on the North Icelandic Shelf well. Near surface-dwelling foraminifera trace anthropogenic CO2 in the Iceland Sea by ~ 1950 ± 8, however, a reduced amplitude shift in the Marine Suess effect is identified. We argue that this is caused by a contemporary ongoing increase in marine primary productivity in the upper ocean due to enhanced Greenland's freshwater discharge that has contributed to a nutrient-driven fertilization since the 1940s/50s (Perner et al., 2019). Multi-decadal variability is detected. We find that the 16-year periodicity evident in SPMW and AIWs based on the δ18O of N. pachyderma and M. barleeanum is a signal of SST anomalies propagated into the Nordic Seas via the Atlantic inflow branches around Iceland. Spectral analyses of the planktic foraminiferal δ13C signal indicate intermittent 30-year cycles that are likely reflecting the ocean response to atmospheric variability, presumably the East Atlantic Pattern. A long-term trend in benthic δ18O suggests that Atlantic-derived waters are expanding their core within the water column from the subsurface into deeper intermediate depths towards the present day. This is a result of increased transport by the North Icelandic Irminger Current to the North Iceland Shelf over the historical era.

Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms.

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.