Small GTPase Signalling
The small GTPase K-Ras is one of the most frequently mutated oncogene products, with activating mutations most prevalent in human pancreatic (95%), thyroid (55%), colorectal (35%), and lung (35%) carcinomas. We intend to identify and characterize druggable target molecules that play a critical role in the Ras signalling pathway in cancer cells that are “addicted” to K-Ras activating mutations (for instance, G12V). The Ras signalling pathway involves many proteins, some of which may offer targets for synthetic lethality with K-Ras mutations in cancer cells. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) are obvious candidates for therapeutic targets, as they directly alter the activity of small GTPase proteins and hence effector proteins downstream in the signalling pathway. By disrupting or inhibiting one of those signalling proteins, it may be possible to specifically kill the oncogene-addicted cells by virtue of “synthetic lethality”. We believe that molecular and structural studies of these key signalling molecules are extremely important to better understand how the signalling pathways in certain cancers are “wired” for cell survival, and how we could exploit cancer-specific molecular pathways to selectively kill tumour cells.
We have recently developed a new real-time NMR methodology to assay small GTPase nucleotide exchange and hydrolysis, as well as the catalysis of these reactions by GEFs and GAPs, respectively. This assay uses the natural ligands GTP and GDP and has been successfully applied to a number of small GTPases including Ras, RhoA, and Rheb. Rheb, a member of the Ras sub-family of small GTPases, functions as an activator of mammalian Target of Rapamycin (mTOR), which is implicated in cell growth, proliferation, and tumourgenesis. We have characterized a number of mutations in the tuberous sclerosis complex 2 (TSC2) protein, a Rheb-specific GAP implicated in the congenital genetic disorder TSC in humans. Using NMR spectroscopy and X-ray crystallography, we are currently investigating regulatory mechanisms of several small GTPases such as Rheb, Ras and RhoA, and addressing questions of how somatic mutations found in GTPases or their GAPs/GEFs deregulate the GTPase cycle, thereby affecting normal cell function and leading to tumourgenesis. Examples include the RhoGEFs, PDZRhoGEF, and Lfc/GEF-H1, as well as RasGAPs and RasGEFs. Our NMR-based GTPase assays could offer a highly specific and reliable tool to perform functional assays for biomarkers and drug development.
Smith MJ, Ottoni E, Ishiyama N,GoudreaultM, Haman A, Meyer A, Tucholska A, Gasmi-SeabrookG, Menezes S, Laister RC, MindenMD, Marschalek R, Gingras A-C, HoangT, IkuraM Evolution of AF6-RAS Association and its Implication in Mixed-Lineage Leukemia. (2017) Nature Comm. 23; 8(1): 1099.
Spencer-Smith R, Koide A, Zhou Y, Eguchi R, Sha F, Gajwani P, Santana D, Gupta A, Jacobs M, Herrero-Garcia E, Cobbert J, Lavoieh H, Smith M, Rajakulendran T, Dowdell E, Okur MN, Dementieva I, Sicheri F, Therrien M, Hancock JF, Ikura M, Koide S, O’Bryan JP. Inhibition of Ras function through targeting an allosteric regulatory site. (2017) Nature Chem Biol 13(1); 62-68.
Mazhab-Jafari MT, Marshall CB, Smith MJ, Gasmi-Seabrook GM, Stathopulos PB, Inagaki F, Kay LE, Neel BG, Ikura M. Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site. (2015) Proc Natl Acad Sci U S A. 112(21):6625-30.
Cullis J, Meiri D, Sandi MJ, Radulovich N, Kent OA, Medrano M, Mokady D, Normand J, Larose J, Marcotte R, Marshall CB, Ikura M, Ketela T, Moffat J, Neel BG, Gingras AC, Tsao MS, Rottapel R The RhoGEF GEF-H1 Is Required for Oncogenic RAS Signaling via KSR-1. (2014) Cancer Cell. 25(2):181-95.
Findlay GM, Smith MJ, Lanner F, Hsiung MS, Gish GD, Kaneko T, Huang H, Bagshaw RD, Ketela T, Moffat J, Ikura M, Li S, Sidhu S, Rossant J, Pawson T. The binding properties of the RasGEF Sos1/Grb2 complex define timing and selectivity in embryonic stem cell lineage commitment. (2013) Cell 152(5):1008-20.
Smith MJ, Hardy WR, Li GY, Goudreault M, Hersch S, Metalnikov P, Starostine A, Pawson T, Ikura M. The PTB domain of ShcA couples receptor activation to the cytoskeletal regulator IQGAP1. (2010) EMBO J. 29(5):884-96.
Marshall CB, Ho J, Buerger C, Plevin MJ, Li GY, Li Z, Ikura M, Stambolic V. Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR. (2009) Science Signal. 2(55):ra3, 1-11.