Elucidating the role of JAK/STAT signalling in polycythaemia vera and essential thrombocythaemia using proteomics

Grayson, Amy Kathryn (2018) Elucidating the role of JAK/STAT signalling in polycythaemia vera and essential thrombocythaemia using proteomics. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Myeloproliferative neoplasms (MPNs) are hyperproliferative disorders affecting the number of circulating cells within the peripheral blood. Previous research has identified the same JAK2 point mutation in three different MPNs; polycythaemia vera (PV), essential thrombocythaemia (ET) and myelofibrosis (MF). The JAK2V617F mutation is thought to be the driver of malignant transformation however, elucidating the mechanisms in which one mutation can result in three different cancers is still a challenge to researchers. The JAK1/2 inhibitor ruxolitinib has been successfully used to alleviate disease-related symptoms and reduce splenomegaly in MPN patients. Furthermore, it has been suggested that STAT1, a direct downstream target of JAK2 activation, is the genetic modifier involved in the development of the malignant phenotypes, with increased STAT1 activity producing an ET-like phenotype, whereas lower STAT1 activity produces a PV-like phenotype. This report will investigate the effect of JAK2 inhibition by ruxolitinib on cellular proliferation and expression of downstream JAK/STAT targets in PV and ET representative cell lines (SET2 and HEL respectively. Cells were treated with 1 μM ruxolitinib and proliferation and cell viability were assessed by growth curve analysis, MTS and Trypan Blue assays. Mass spectrometry was used to identify molecules of significance with respect to JAK2 signalling in ET through differential expression upon ruxolitinib treatment. Following this, Western Blot and Q-RT-PCR was performed on molecules of interest. Ruxolitinib was also investigated as a dual agent alongside IFN-α, a STAT1 activator, and fludarabine, a STAT1 inhibitor, to investigate the specific role of STAT1 in both PV and ET and possible synergistic effects of two treatments currently used with MPN patients. Results show that ruxolitinib had a similar effect on cell proliferation and viability in both SET2 and HEL cells. Protein and mRNA expression of known JAK2 downstream signalling targets were shown to be downregulated in response to ruxolitinib treatment in SET2 cells and a selection of molecular chaperone genes, transcription factors, cell cycle regulators and genes involved in antigen presentation were affected by ruxolitinib treatment. Treatment of SET2 and HEL cells with a combination of IFN-α and ruxolitinib showed a similar pattern as treatment with ruxolitinib alone with respect to cell proliferation and viability, suggesting this combination may not be the most effective treatment for PV and ET. Combination treatment of fludarabine and ruxolitinib resulted in a much greater decrease in proliferation and viability in both cell lines compared to ruxolitinib treatment alone, proposing a new potential treatment combination for this subset of MPNs.