Abstract
Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). Of these, approximately half are classified as type 1 and 30% as type 2, characterized by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5) respectively. Although both share identical mutant C-termini and are able to bind and activate MPL, type 1 and type 2 CALR mutations display different clinical and prognostic presentation: type 1 mutations are associated primarily with a fibrotic phenotype and increased proclivity towards fibrotic transformation, while type 2 mutations are more common in ET. Molecularly, type 1 and type 2 mutations result in differential C-domain amino acid sequences with the potential to affect the function of the protein. Various well known functions of CALR, including calcium binding ability and protein folding capacity, have begun to be explored in the context of CALR mutations; however, the impact of CALR mutations on its acetyltransferase ability, which was only discovered in 2006, remains unknown.
Here, we show that in accordance with our structural models, mutant CALR not only retains its acyltransferase ability, but type 1 CALRdel52 mutations specifically lead to increased activation of its acetyltransferase ability, revealing a new gain of function phenotype for CALRdel52 mutations. As a result, type 1 CALR mutations lead to increased acetylation of CALR's acetyltransferase targets downstream, such as glutathione-S-transferase and cytochrome P450 reductase, which affects the outputs of these pathways downstream. Exploratory RNA-Seq on CALR-mutated cells revealed a concurrent upregulation of transferrin receptor mediated iron metabolism by CALRdel52. We subsequently validated this finding and show that CALRdel52 cells display differential iron metabolism.
Given the upregulation of the transferrin receptor and the increased acetyltransferase ability affecting proteins involved in reactive oxygen species pathways (ROS), ferroptosis-an iron-dependent form of cell death characterized by the accumulation of lipid peroxides-emerged as a potential therapeutic target for CALRdel52 mutated cells. To test this, we first assessed basal proclivity to ferroptosis by measuring the lipid peroxidation product, classic ferroptotic marker 4-HNE (4-hydroxynonenal) as well as both ROS and global lipid peroxide levels in cells expressing wild-type CALR, CALRdel52, and CALRins5. We found that all of these ferroptotic markers were significantly increased in CALRdel52 cells. Therapeutic modulation of these pathways such as iron supplementation resulted in targeting of CALRdel52 cells and ferroptosis induction. This work is the first to examine the acetyltransferase ability of mutant CALR and reveal downstream phenotypic differences based on this ability that set the groundwork for a host of unexplored cellular consequences. Moreover, this study unites the novel understanding of the acetyltransferase function of mutant CALR with changes in transferrin receptor mediated iron metabolism to reveal not only how CALRdel52 induces a ferroptotic proclivity, but the potential of this sensitivity for therapeutic targeting.
No relevant conflicts of interest to declare.
