Phosphoproteomic investigation of kinase signalling network plasticity in response to chronic PI3K and mTORC1/2 inhibition
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Class I phosphoinositide 3-kinases (PI3K) and mammalian target of rapamycin complexes 1/2 (mTORC1/2) are enzymes that play important roles in elementary biology and disease. As a consequence, numerous small-molecule inhibitors of their catalytic activity have been developed and these have shown clinical utility in certain cancers. Unfortunately, acquired resistance to these therapies is a common phenomenon and often occurs relatively quickly following treatment. Our understanding of how resistance develops is hampered by the difficulty of measuring the circuitry and plasticity of the signalling networks that these and other kinases signal within. Advances in mass spectrometric technologies have rendered the routine quantitative interrogation of the phosphoproteome (the set of phosphorylated proteins expressed in a particular biological system at a specific time) more tractable than ever before. The aim of this project therefore, was to improve upon existing mass spectrometry (MS)-based phosphoproteomics methods, and to utilise these to contribute to our understanding of kinase signalling networks and examine their plasticity in models of acquired resistance to PI3K and mTORC1/2-targeted therapies. Novel approaches for the enrichment of phosphopeptides from complex biological matrices (and their analysis by MS) were designed, tested and optimised. These methods were then used to systematically characterise a kinase signalling network comprising the Akt/PI3K/mTOR and MEK/ERK signalling axes in MCF7 breast cancer cells. The biological relevance of this network was confirmed through the assessment of its dynamics upon EGF and IGF-1 stimulation. Finally, the plasticity of this network following chronic treatment with targeted PI3K and mTORC1/2 inhibitors (GDC-0941 and KU-0063794) was examined in cell-line models of acquired resistance to these two compounds. This revealed that these cells each remodelled this network in a different manner, thus indicating that the initial conditions of the system were not the sole determinant of how resistance was acquired.
AuthorsWILKES, Edmund H
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