Proposal Summary/Abstract Myeloproliferative Neoplasm (MPN) is a group of chronic blood disorders. The majority of MPN patients have acquired a JAK2V617F mutation which leads to constitutive signalling of myeloid growth factor receptors resulting in excessive production of mature myeloid cells. MPN patients have additionally exhibited dampened IL-10R signalling responses that has shown to give the JAK2V617F mutant cells a competitive advantage for outgrowth. This blunted IL-10 response is linked to increased TNF-a production, leading to persistence of inflammation and proliferative stress in non-mutant HSCs resulting in HSC exhaustion. As JAK2 canonically does not participate in the IL-10R pathway, the mechanism by which JAK2V617F mutant cells rescue this pathway is still unknown. We hypothesise that mutant JAK2V617F is able to utilise this transactivation mechanism and trans- activate JAK1 in order to induce signalling via the IL-10R pathway, allowing the JAK2V617F selective advantage in MPN dampened IL-10 signalling. In understanding the mechanism JAK2V617F positive cells are able to use to take advantage of the dysfunctional IL-10 response seen in MPN patients, may open up a new therapeutic target as to not only fight against disease progression but also to take away the aforementioned advantage of the mutant JAK2V617F. A key component of our study is the use of a murine pro B-cell, Ba/F3, that is cytokine dependent as a means of not just analysing cell signalling but to also observe the transformative ability of the JAK2V617F mutant. Aim 1 we want to identify the mechanism of JAK2V617F’s non-canonical IL-10R signalling. Both Western blotting and Co-immunoprecipitation techniques will be used to analyse JAK-STAT signalling and hypothesised non- canon JAK1/JAK2 heterodimerisation under IL-10 stimulation and in the absence of cytokine. For Aim 2 we want to determine JAK1’s dependency, role and how it mediates this signalling pathway. To study this siRNAs against mJAK1 will be developed and used, followed by western blot analysis in order to study the JAK-STAT signalling occurring under IL-10 stimulation as previous, but to also determine decrease in JAK1 expression. Aim 3 will be identifying means to inhibit this non-canonical signalling. JAK2 inhibitors, like Pacritinib, will be used as a means to potentially ablate the transactivation mechanism of JAK2V617F and to return the IL-10R signalling back to normalcy.