Alterations of Signaling Pathways in Essential Thrombocythemia with Calreticulin Mutation

Somatic mutation of calreticulin (CALR) has been found in about 25% patients with essential thrombocythemia (ET), where is the second most common mutated gene after JAK2V617F.Recent data have shown that thrombopoietin receptor MPL function is essential in mutant CALR-mediated cellular transformation. However, the mechanisms for CALR mutant-driven MPN have not been fully elucidated.

The goal of this study is to characterize dysregulation of protein expression in ET patients with CALR mutation using Protein Pathway Array (PPA) and to understand possible involvement of signaling pathways related to CALR mutation.

This study included 18 ET patients with CALR mutation and 20 ET patients with JAK2V617F mutation and 20 healthy controls. Total proteins were extracted from peripheral blood neutrophil of samples. The expression of 128 proteins was assessed using PPA.PPA is high-throughput proteomic assay, which combines multiplex immunoblot with computational analysis.

There were 20 proteins were found to be dysregulated in ET patients with CALR mutation compared with those in healthy controls (p<0.05). Among them, 13 proteins including Erk1/2, PTEN, Rap1, Raf-B, Axin, Cdc42, eIF4B, Bcl-Xl, c-IAP2, NFκB p50, TGF-β, CyclinD1 and SRC-1 were up-regulated in ET patients, while 7 proteins including cPKCα, TDP1, Cdc2, ERβ, Survivin, E-cadherin and p27 were down-regulated in ET patients.

To identify the significant pathways affected by these differential expression of the proteins, we analyzed these 20 proteins using Ingenuity Pathways Analysis(IPA) and top 10 canonical pathways were identified. These pathways included PTEN signaling, apoptosis signaling,IL-8 signaling,PI3K-AKT signaling,regulation of the epithelial-mesenchymal transition pathway,ILK signaling,protein kinase A signaling,HGF signaling,IL-12 signaling,cyclins and cell cycle regulation. These results suggest cellular proliferation,apoptosis and cytokine pathways may be involved in CALR mutant-driven ET.

To better understand the protein-protein interaction network as well as the major regulators, 20 dysregulated proteins identified by PPA were imported to the IPA and the signal network was built. In this network, core regulators include PTEN,CyclinA, CyclinD1, Erk1/2,E-cadherin,p27.These core regulators may play an important role in pathogenesis of ET with CALR mutation.

Furthermore, our results showed a significant overlap in the protein expression patterns between ET patients with CALR mutation and with JAK2V617F mutation. To determine the difference in activated signaling pathways between ET patients with CALR mutation and with JAK2V617F mutation, the protein expression level between these 2 groups were compared and 8 proteins(Erk1/2, TGF-β, CyclinD1, Stat5 , p27, IL-1β, ASCL, HIF1α ) were found to be differentially expressed (p<0.05). These proteins might relate to the signaling pathway of activation by CALR mutation, which was different to JAK-STAT signaling by JAK2V617F mutation.

In conclusions, our study suggests that a broad dysregulation of signaling proteins in ET patients with CALR mutation. These findings increase our understanding of CALR mutant characteristics and offer insights into the mechanisms of ET.