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

项目目标：a)确定哪一个Tetraspanin/ADAM10剪刀(S)激活T-ALL中的Notch1)研究Notch1是在细胞表面还是在内化后的小泡上被切割C)确定针对Notch1的剪刀是否抑制T-ALL的增殖A)确定哪一个Tetraspanin/ADAM10剪刀(S)激活T-ALL中的Notch1我们将使用我们展示的Jurkat模型T-ALL细胞系表达Tspan5、Tspan14、Tspan15、Tspan17和Tspan3311。我们将使用CRISPR/Cas9敲除单个TspanC8，并使用转录荧光素酶报告基因分析来测量Notch1的活性。在Jurkat细胞中的研究结果将扩展到具有不同类别Notch1激活突变的T-ALL细胞，如KOPTK1和P12 Ichikawa细胞。B)为了研究Notch1是在细胞表面还是在内化后的小泡上被切割我们将使用共聚焦和结构照明成像，以及荧光相关光谱，来对转染荧光标记的TspanC8s、ADAM10和Notch1切割传感器进行成像，以确定发生配体无关的突变Notch1激活的位置。C)确定是否靶向Notch1抑制位T-ALL增殖我们显示了在ADAM10基因敲除后Jurkat细胞(图1B)的增殖减少，这与已发表的数据14一致。此外，结合Tspan14基因敲除和Tspan5抗体可以抑制配体诱导的Notch1信号。我们将测试TspanC8基因敲除或TspanC8抗体治疗是否会抑制T-ALL细胞系和原代细胞的增殖。我们最近已经产生了Tspan14和Tspan15的单抗(Tomlinson实验室，未发表)。我们将与业界合作生产更多抗体；UCB Pharma外部发现解决方案总监Tom Crabbe博士向我们提供了UCB的噬菌体展示抗体技术。

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 We have recently generated Tspan14 and Tspan15 monoclonal antibodies (Tomlinson lab, unpublished).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.