| Summary: | The TeIjPDGF~R {TP} chimeric protein is an activated tyrosine kinase, implicated in the pathogenesis of chronic myelomonocytic leukaemia {CMML}. In order to investigate in more detail the coupling of TP induced molecular signals to functional responses relating to haemopoiesis, we have used the tetracycline-regulated expression system in murine ES cells. The use of this system provides an ideal in vitro model to monitor the effects of expressing TP not only in primitive stem cells with pluripotent potential but also during the initiating and more definitive stages of haemopoiesis. Results indicate that expression of TP results in marked increase in differentiation of ES cells, decreasing the overall levels of transcription factors associated with pluripotency whilst increasing the expression of factors known to haemopoietic differentiation of ES cells TP promotes differentiation along the mesodermaljhaemangioblast/myelomonocytic route, resulting in a marked increase in myelopoiesis with on average 20% more myeloid colonies forming. TP also activates several major signalling pathways with tyrosines 579/581 playing a critical role in mediating signals via the RAS/ERK and STAT5 pathways, with dual targeting of the tyrosine kinase activity of TP and the MEK/ERK pathway completely preventing TP ind uced differentiation. Although activation of the Wnt/GSK3~/~- catenin pathway has been illustrated in other myeloid leukaemia's, in our study TP down regulated this pathway in our stem cell model. Activation of the pathway using three independent pharmacological compounds, supplementation with Wnt3a and expressing a dominant positive form of ~-catenin improved self-renewal v and reversed transcriptional changes induced following TP induction. Overall this study highlights an important role for the RAS/ERK, STATS and Wnt/GSK313/13- catenin pathways in leukaemic transformation by TP, and reinforces the principle that targeted disruption of key signalling pathways alongside tyrosine kinase oncogenes may result in more efficacious therapies for suppressing MPN.
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