SOCS3 Phosphorylation In Myeloproliferative Neoplasms

Suppressor of cytokine signaling protein 3 (SOCS3) is an important regulator of the erythropoietin receptor and the receptor-associated JAK2 kinase. We hypothesize that the mechanism of JAK2 mutant-mediated transformation in myeloproliferative neoplasms (MPN) involves overcoming SOCS3 inhibition. In co-expression studies SOCS3 protein is stabilized and tyrosine phosphorylated in the presence of JAK2 V617F (Hookham et al., Blood 2007) and exon 12 mutations (Elliott et al., Haematologica 2009) but not wild-type JAK2, rendering SOCS3 unable to downregulate phosphorylation of these JAK2 mutants. The purpose of this study was to determine if these observations can be reproduced in vivo, by comparing SOCS3 expression and phosphorylation in MPN patients and healthy volunteers.

Patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) were recruited from Belfast City Hospital. The mutational status of the JAK2 gene had been previously assessed. Granulocytes were isolated from peripheral blood of patients and healthy volunteers by Ficoll density gradient centrifugation, dextran sedimentation and hypotonic red cell lysis. Estimation of the JAK2 V617F/wild type allelic ratio was performed by pyrosequencing. SOCS3 expression levels were measured by quantitative real-time reverse transcriptase-PCR. Granulocyte whole cell lysate was immunoprecipitated with anti-SOCS3 and Western blots were probed with anti-phosphotyrosine. SOCS3 phosphorylation was estimated by densitometry.

To date, 63 MPN patients and 27 healthy volunteers have been recruited. This total is comprised of 47 patients with PV (JAK2 V617F n=38, JAK2 exon 12 n=4, unknown n=5) and 16 patients with ET (JAK2 V617F n=15). Median V617F allele burden was 45% in PV (range 0–99%) and 20% in ET (range 7–84%). In 67% of patients with PV, SOCS3 expression was greater than healthy volunteers. Compared to healthy volunteers, SOCS3 phosphorylation in PV patients was higher in 67%, equivalent in 17% and lower in 17% of cases. Similarly in ET, SOCS3 phosphorylation was higher in 73%, equivalent in 18% and lower in 9% of patients compared with healthy volunteers. SOCS3 tyrosine phosphorylation was observed in association with both JAK2 V617F and exon 12 mutants.

The presence of aberrant tyrosine phosphorylation of SOCS3 protein observed in some PV and ET patients supports the hypothesis that this is a potential mechanism by which mutant JAK2 escapes inhibition and leads to constitutive downstream signaling in MPN.

No relevant conflicts of interest to declare.