Understanding how tetraspanins and the 'molecular scissor' ADAM10 promote blood cancer

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Project aims:A) To determine which tetraspanin/ADAM10 scissor(s) activates Notch1 in T-ALLB) To investigate whether Notch1 is cut at the cell surface or on vesicles following internalisationC) To determine whether targeting the scissor for Notch1 inhibits T-ALL proliferationA) To determine which tetraspanin/ADAM10 scissor(s) activates Notch1 in T-ALLWe will use the Jurkat model T-ALL cell line that we showed to express Tspan5, Tspan14, Tspan15, Tspan17 and Tspan3311. We will knock out individual TspanC8s using CRISPR/Cas9 and measure Notch1 activity using a transcriptional luciferase reporter assay. Findings in Jurkat cells will be extended to T-ALL cells with different classes of Notch1-activating mutations, such as KOPTK1 and P12 ICHIKAWA cells.B) To investigate whether Notch1 is cut at the cell surface or on vesicles following internalisationWe will use confocal and structured illumination imaging, together with fluorescence correlation spectroscopy, to image transfected fluorescent-tagged TspanC8s, ADAM10 and a Notch1 cleavage-sensor to determine where ligand-independent mutant Notch1 activation occurs.C) To determine whether targeting the scissor for Notch1 inhibits T-ALL proliferationWe have shown reduced proliferation following ADAM10 knockout in Jurkat cells (Figure 1B), consistent with published data14. Moreover, combined Tspan14 knockdown and a Tspan5 antibody inhibits ligand-induced Notch1 signalling9. We will test whether knockout of TspanC8s, or treatment with TspanC8 antibodies, inhibits proliferation of T-ALL cell lines and primary cells. We have recently generated Tspan14 and Tspan15 monoclonal antibodies (Tomlinson lab, unpublished). We will partner with industry to generate additional antibodies; Dr Tom Crabbe, Director of External Discovery Solutions at UCB Pharma, has offered us UCB's phage-display antibody technology.