In vivo analysis of the specificity of outputs of receptor tyrosine kinase signalling by high content microscopy

This project is supervised by Dr R. Kelsh, Dr J. Caunt and and Dr S. Ward

Project description:

Receptor tyrosine kinases are a major class of transmembrane receptors, with diverse roles in development and disease, e.g. cancer. The precise function of each kinase is distinct, yet the intracellular signalling pathways that mediate those signals are shared between all the cells. It remains a major challenge to explain how these shared pathways generate distinct outputs in a cell- and kinase-specific way. One such kinase, Anaplastic Lymphoma Kinase (ALK) is constitutively activated in multiple distinct cancer types, including non-small cell lung cancer, anaplastic large cell lymphoma and neuroblastoma. In many cases, constitutive activation comes about by chromosomal translocations which resulting in chimaeric fusion proteins, e.g. NPM-ALK. We have developed a novel in vivo assay for ALK activity in the zebrafish; this assay is highly visual, with pigment cell number being the readout (Rodrigues et al, 2012 ACS Chem Biol, accessible online). We see a similar effect from an analogous construct where we substitute the ALK intracellular (kinase) domain with that from the sister kinase Ltk, consistent with the endogenous role of Ltk in the zebrafish (Lopes et al., 2008, PLoS Genetics 4, e1000026). In contrast, another RTK, Kit, has a very different endogenous function (melanocyte migration and survival). By engineering a Kit-based construct, we will now assess whether this more divergent kinase generates a similar or distinct phenotypic output. Whether the response in this artificial assay is similar or distinct, we will now have a system in which to dissect the molecular basis for the specificity of response from these RTKs. One important possibility is that the specificity results from crucial, but perhaps subtle, differences in the combinations of distinct secondary signalling outputs (PI3K, MEK/ERK, JAK/STAT) stimulated by each ALK fusion. We have recently developed fluorescent live reporters for these signalling pathways and have shown their suitability for High Content Microscopy (HCM). By combining these novel tools with the in vivo and more conventional cell culture (in vitro) assays, and using a state-of-the-art HCM system, the successful candidate will analyse the effects of different RTK proteins on both pigment cell development and the outputs of each of the secondary signalling pathways. This will allow correlation of the relationship between the balance of signalling outputs and the cellular outcome. In order to generate a comprehensive understanding of how different RTK proteins generate distinct outcomes, we wil use highly specific chemical and genetically-engineered inhibitors of the individual secondary signalling systems. Together, these studies will provide detailed insights into the crucial problem of how receptor tyrosine kinase signalling has profoundly different effects in a cell-type and receptor-type specific manner.