Transcriptomic Analysis Reveals JAK2/MPL-Independent Effects of Calreticulin Mutations in a C. elegans Model

Abstract

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.

CALR but Not JAK2 Mutations Are Associated with an Overexpression of Retinoid X Receptor Alpha in Essential Thrombocythemia

“…Thus, all the effects generated by the introduction of the mutated calreticulin in this model should be a consequence of mechanisms that are independent of JAK2/STAT. In previous studies, we observed that the introduction of patient-like mutations in the calreticulin of C. elegans led to an increase in the transcriptional expression of nhr-2 (Nuclear Hormone Receptor 2), which seems to be involved in transcription regulation and cell differentiation [2]. Based on these findings, we searched for possible human orthologous genes of nhr-2 to determine if an increase in their expression was also derived from mutant calreticulin in patients with ET, which would point to new potential therapeutic targets in these patients.…”