Investigation of JAK2 targets in haematological malignancy

Janus kinase 2 (JAK2) is a critical activator of signalling associated with many different receptors, particularly those associated with haematopoiesis and the inflammatory response. Classically JAK2 has been thought of as a cytoplasmic protein, but more recently there is evidence that JAK2 is able to enter the nucleus leading to alteration of epigenetic modifiers and enhancement of JAK2 transcriptional targets. This role in haematopoiesis means that alteration of JAK2 signalling, such as in malignant haematopoiesis, can be a critical factor in the development and maintenance of disease. The myeloproliferative neoplasms (MPN) are a group of conditions characterised by over-production of myeloid cells as a result of acquired mutations in haematopoietic stem cells. Two well-known MPNs are chronic myeloid leukaemia (CML), driven by the novel oncoprotein BCR-ABL, and polycythaemia vera (PV), driven by the JAK2 V617F activating mutation. JAK2 is critical for normal haematopoiesis through its role in initiating signalling following activation of a receptor by its ligand. In this study we looked several aspects of JAK2 activity, including its effect on gene expression, its activity within the nucleus, and the effect of overactivity on aspects of inflammation. Here we show that gene expression in both CML and PV patient samples is deregulated compared to normal controls with both previously confirmed alterations such as BCL2 and MCL1 in CML and STAT1 and MPL in PV, and novel alterations such as Rex1 and SUMO2 in CML and EGR1 in PV. These alterations at the level of mRNA transcription are validated by significant alterations in cell cycle and apoptosis in cell lines following treatment with JAK2 inhibitors. As part of the investigation of the activity of JAK2 in the nucleus we hypothesised that JAK2 may interact directly with PML nuclear bodies, a subnuclear structure with numerous regulatory roles in apoptosis, cell cycle, DNA repair and response to infection. Using immunofluorescence based techniques in cell lines and patient samples we showed that JAK2 does interact directly with PML nuclear bodies. This interaction was affected by JAK2 inhibitors in cell lines and increased in PV patient monocytes compared to normal controls. Treatment of cell lines with the JAK2 inhibitor ruxolitinib and arsenic trioxide, which degrades PML, led to an increase in apoptosis compared to either compound alone, suggesting a meaningful functional interaction in both normal and malignant haematopoiesis. The role of the JAK2 V617F in cellular function has been extensively investigated particularly in neutrophils and platelets. However the effect of the JAK2 V617F mutation on monocytes and monocyte derived macrophages has not previously been investigated. Here we show that patients with PV have an alteration in monocyte subsets with an increase in the inflammatory intermediate monocytes at the expense of classical monocytes. We also showed that macrophages derived from peripheral blood monocytes obtained from patients with JAK2 V617F positive PV behaved differently from those derived from normal controls. There were alterations both at rest and in response to stimulation in terms of the morphology, with a more inflammatory phenotype seen, and in cytokine release with blunting of some responses and heightening of other. These alterations were seen both in M1 and M2 macrophages. Overall the work presented in this thesis shows wide ranging alterations in many aspects of cellular behaviour as a result of overactivity of JAK2. Some of these findings have been previously identified and discussed, but there are several key novel findings which progress out understanding of JAK2 function both in normal and malignant haematopoiesis, and the effects of malignant JAK2 activation on cellular behaviour