Mutations in the CALR gene, including a 52 base pair (bp; CALR Del52) deletion and 5 bp insertion (CALR Ins5), affect several signaling pathways in cells leading to the pathogenesis of myelofibrosis (MF) and other myeloproliferative neoplasms (MPNs), according to a study published in the International Journal of Molecular Sciences.
Cells carrying CALR Del52 and CALR Ins5 mutations had increased activation of the Janus kinase/signal transducer and activator of the transcription (JAK/STAT) and the phosphatidylinositol 3-kinase/Protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathways which have been previously implicated in the pathogenesis of MPNs. These effects were still present in a cell culture model lacking MPL gene (thrombopoietin receptor) expression.
The CALR mutations resulted in reduced functionality of calreticulin proteins. Calreticulin generally functions as a major chaperone in the endoplasmic reticulum and is involved in several processes, including control of protein folding, calcium homeostasis, and responses to cellular stress.
The study found cells with CALR Del52 mutations had statistically significant higher levels of DNA damage compared to controls when exposed to hydrogen peroxide. Cells with CALR Ins5 had significantly higher levels of phosphorylated ATM and H2AX than controls. Both cell types were not able to repair DNA damage after 24 hours following oxidative stress.
Apoptosis levels were also significantly higher in cells with the CALR Ins5 mutation compared to controls. Those with CALR Del52 also had higher rates of apoptosis, but it did not reach significance. Further analysis found that the CALR mutations not only led to increased apoptosis after hydrogen peroxide exposure-induced oxidative stress but also tended to arrest the cells in the G2/M phase.
“Functional analysis revealed that the accumulation of G2/M-CALR-mutated cells indicates that oxidative stress-induced DNA damage is difficult to repair. The mentioned cell cycle delay has not been shown in other studies analyzing mutated calreticulin,” the authors said.