Calreticulin Ins5 and Del52 Mutations Impair Unfolded Protein and Oxidative Stress Responses in Hematopoietic Cells

Somatic mutations of calreticulin (CALR) have been described in approximately 30-40% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In order to assess whether and how CALR mutations could affect the physiological CALR protein functions in the ER and thus contributing through other mechanisms to the development of MPNs, we decided to study the role of mutated CALR in K562 cells, devoid of MPL expression. To this end, K562 cells stably expressing either wt CALR or the two most common CALR mutated variants CALRdel52 and CALRins5 were generated via retroviral mediated gene transfer.

To identify common signalling pathways modulated by CALR mutants, GEP analysis was performed. Ingenuity Pathway analysis performed on Differentially Expressed Gene (DEGs) revealed that the categories "Unfolded protein response", "Endoplasmic Reticulum Stress Pathway", and "NRF2-mediated Oxidative Stress Response" were significantly represented in the list of decreased genes in the comparison mutated vs wt K562. Based on these findings, the ability to respond to ER and oxidative stresses were assessed in K562 carrying either wt or mutated CALR. Our data demonstrated that CALR mutants negatively impact on the Unfolded Protein Response (UPR): in particular, CALR mutations appear to reduce the activation of the pro-apoptotic pathway downstream the UPR, therefore allowing the accumulation of misfolded proteins in the ER and conferring resistance to ER stress-induced apoptosis. Moreover, our results showed that CALR mutations also affect the capability to respond to oxidative stress: K562 cells carrying CALR mutants showed decreased SOD activity coupled to increased ROS intracellular levels, suggesting that CALR mutants impair cell ability to counteract ROS accumulation. Furthermore, cells carrying CALR mutants showed increased levels of DNA damage upon oxidative stress exposure and decreased ability to repair the oxidative DNA damage. We also demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR.

Altogether our data suggest a novel MPL-independent role for CALR mutations in the development of MPNs. On one side, by affecting the ability to respond to the ER stress, CALR mutants confer resistance to ER stress mediated apoptosis. On the other side, by affecting cell sensitivity to oxidative stress and reducing the capability to respond to oxidative DNA damage, CALR mutants might lead to genomic instability and tendency to accumulate further mutations.