Uncoupling TORC2 from AGC kinases inhibits tumour growth

// Angus J.M. Cameron 1 , Selvaraju Veeriah 2,3 , Jacqueline J.T. Marshall 3 , Elizabeth R. Murray 1 , Banafshe Larijani 4 and Peter J. Parker 3,5 1 Kinase Biology Laboratory, Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London, UK 2 Translational Cancer Therapeutics Laboratory, Paul O’Gorman Building, University College London Cancer Institute, London, United Kingdom 3 Protein Phosphorylation Laboratory, Francis Crick Institute, London, UK 4 Cell Biophysics Laboratory, Ikerbasque Basque Foundation for Science, Research Centre for Experimental Marine Biology and Biotechnology (PiE) & Biofisika Instituto (UPV/EHU, CSIC), University of the Basque Country, Areatza Hiribidea, Plentzia, Spain 5 Division of Cancer Studies, King’s College London, New Hunts House, Guy’s Campus, London, UK Correspondence to: Angus J.M. Cameron, email: // Peter J. Parker, email: // Keywords : mTORC2, CRIM, Akt, xenograft, Sin1 Received : December 29, 2016 Accepted : June 26, 2017 Published : August 09, 2017 Abstract Mammalian target of rapamycin (mTOR) is a central regulator of growth and metabolism. mTOR resides in two distinct multi-protein complexes – mTORC1 and mTORC2 – with distinct upstream regulators and downstream targets. While it is possible to specifically inhibit mTORC1 with rapamycin, or inhibit both mTOR complexes together with ATP pocket directed mTOR kinase inhibitors, it is not possible to assess the specific roles for mTORC2 pharmacologically. To overcome this, we have developed a novel, inducible, dominant negative system for disrupting substrate recruitment to mTORC2. Previously we identified the mTORC2 specific subunit Sin1 as a direct binding partner for AGC kinases Akt and PKC. Sin1 mutants, which retain the ability to bind Rictor and mTOR, but fail to recruit their AGC client kinases, inhibit AKT and PKC priming and block cell growth. In this study, we demonstrate that uncoupling mTORC2 from AGC kinases in DLD1 colon cancer cells inhibits Akt activation and blocks tumour growth in vivo . Further we demonstrate, using time resolved two-site amplified FRET (A-FRET) analysis of xenograft tumours, that inhibition of tumour growth correlates with the degree of mTORC2 uncoupling from its downstream targets, as demonstrated for Akt. These data add weight to the body of evidence that mTORC2 represents a pharmacological target in cancer independently of mTORC1.