Subject(s)
Leukemia, Myeloid, Acute/blood , Leukemia, Myeloid, Acute/genetics , Oxidation-Reduction , Adult , Aged , Antimycin A/pharmacology , Antioxidants/metabolism , Female , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/mortality , Lipid Peroxidation , Male , Middle Aged , Mitochondria/metabolism , NADPH Oxidases/metabolism , Onium Compounds/pharmacology , Prognosis , Proportional Hazards Models , Prospective Studies , Reactive Oxygen Species/metabolism , Rotenone/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Translocation, GeneticABSTRACT
Apart from well-known genetic abnormalities, several studies have reported variations in protein expression in Philadelphia-negative myeloproliferative neoplasm (MPN) patients that could contribute toward their clinical phenotype. In this context, a quantitative mass spectrometry proteomics protocol was used to identify differences in the granulocyte proteome with the goal to characterize the pathogenic role of aberrant protein expression in MPNs. LC/MS-MS (LTQ Orbitrap) coupled to iTRAQ labeling showed significant and quantitative differences in protein content among various MPN subtypes [polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)], and according to the genetic status of JAK2 (JAK2V617F presence and JAK2V617F allele burden). A number of differentially expressed proteins were identified, with the most frequent being members of the RAS GTPase family and oxidative stress regulatory proteins. Subsequent analysis found that calreticulin (CALR), known to be involved in calcium homeostasis and apoptotic signaling, was overexpressed in JAK2V617F granulocytes compared with JAK2 wild type and independently of the JAK2V617F allele burden. Finally, it was demonstrated, in a Ba/F3 cell model, that increased calreticulin expression was directly linked to JAK2V617F and could be regulated by JAK2 kinase inhibitors.Implications: In conclusion, these results reveal proteome alterations in MPN granulocytes depending on the phenotype and genotype of patients, highlighting new oncogenic mechanisms associated with JAK2 mutations and overexpression of calreticulin. Mol Cancer Res; 15(7); 852-61. ©2017 AACR.
Subject(s)
Calreticulin/genetics , Janus Kinase 2/genetics , Polycythemia Vera/genetics , Primary Myelofibrosis/genetics , Thrombocythemia, Essential/genetics , Exons , Female , Gene Expression Regulation, Neoplastic/genetics , Genetic Heterogeneity , Genotype , Granulocytes/metabolism , Humans , Male , Mutation , Polycythemia Vera/blood , Polycythemia Vera/pathology , Primary Myelofibrosis/blood , Primary Myelofibrosis/pathology , Proteome/genetics , Thrombocythemia, Essential/blood , Thrombocythemia, Essential/pathologyABSTRACT
Besides genetic abnormalities in MPN patients, several studies have reported alterations in protein expression that could contribute towards the clinical phenotype. However, little is known about protein modifications in Ph- MPN erythrocytes. In this context, we used a quantitative mass spectrometry proteomics approach to study the MPN erythrocyte proteome. LC-MS/MS (LTQ Orbitrap) analysis led to the identification of 51 and 86 overexpressed proteins in Polycythemia Vera and Essential Thrombocythemia respectively, compared with controls. Functional comparison using pathway analysis software showed that the Rho GTPase family signaling pathways were deregulated in MPN patients. In particular, IQGAP1 was significantly overexpressed in MPNs compared with controls. Additionally, Western-blot analysis not only confirmed IQGAP1 overexpression, but also showed that IQGAP1 levels depended on the patient's genotype. Moreover, we found that in JAK2V617F patients IQGAP1 could bind RhoA, Rac1 and Cdc42 and consequently recruit activated GTP-Rac1 and the cytoskeleton motility protein PAK1. In CALR(+) patients, IQGAP1 was not overexpressed but immunoprecipitated with RhoGDI. In JAK2V617F transduced Ba/F3 cells we confirmed JAK2 inhibitor-sensitive overexpression of IQGAP1/PAK1. Altogether, our data demonstrated alterations of IQGAP1/Rho GTPase signaling in MPN erythrocytes dependent on JAK2/CALR status, reinforcing the hypothesis that modifications in erythrocyte signaling pathways participate in Ph- MPN pathogenesis.
