Ph-negative myeloproliferative neoplasms (polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF)) are infrequent blood cancers characterized by signaling aberrations. Shortly after the discovery of the somatic mutations in JAK2, MPL, and CALR that cause these diseases, researchers extensively studied the aberrant functions of their mutant products. In all three cases, the main pathogenic mechanism appears to be the constitutive activation of JAK2/STAT signaling and JAK2-related pathways (MAPK/ERK, PI3K/AKT). However, some other non-canonical aberrant mechanisms derived from mutant JAK2 and CALR have also been described. Moreover, additional somatic mutations have been identified in other genes that affect epigenetic regulation, tumor suppression, transcription regulation, splicing and other signaling pathways, leading to the modification of some disease features and adding a layer of complexity to their molecular pathogenesis. All of these factors have highlighted the wide variety of cellular processes and pathways involved in the pathogenesis of MPNs. This review presents an overview of the complex signaling behind these diseases which could explain, at least in part, their phenotypic heterogeneity.

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Calreticulin (CALR) is a multifunctional calcium-binding protein whose expression levels have been correlated with detection, clinical phase of disease, metastasis, and survival of various types of cancer. Therefore, the study of the regulation of the cellular levels of calreticulin may be important to understand the neoplastic process. Caenorhabditis elegans, which has a CALR ortholog (CRT-1), has been used as a model organism for the characterization of calreticulin, and several conditions promoting the upregulation of crt-1 have been studied and established to understand the molecular control of crt-1 transcription and assess the function of the protein. Here, we propose several modifications of previously published crt-1 upregulation strategies that improve the reproducibility of the assay and allow to achieve higher levels of overexpression. Firstly, the manipulation of synchronized populations of worms instead of mixed-stage animals and the use of solid culture medium in all experimental conditions are proposed. Likewise, we evaluate four new experimental approaches that attempt to promote a higher crt-1 upregulation (short-term exposure to 30 µg/mL tunicamycin at 25 °C, short-term exposure to 7% ethanol at 25 °C, short-term exposure to 30 °C of worms grown at 25 °C, and a long-term exposure to 7% ethanol). Our results not only validate previously published methods, but also point to a new experimental approach that increases previously achieved levels of crt-1 upregulation. More specifically, a 6-hour exposure of synchronized worms grown at 25 °C to 7% ethanol on solid medium promotes almost a 7-fold upregulation of crt-1.

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research.