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

项目目标：A）确定哪种四跨膜蛋白/ADAM 10激酶激活T-ALL中的Notch 1; B）研究Notch 1在内化后是否在细胞表面或囊泡上被切割; C）确定靶向Notch 1的激酶是否抑制T-ALL增殖; A）确定哪种四跨膜蛋白/ADAM 10激酶激活T-ALL中的Notch 1。我们将使用Jurkat模型T-ALL细胞系，我们显示该细胞系表达Tspan 5、Tspan 14、Tspan 15、Tspan 17和Tspan 3311。我们将使用CRISPR/Cas9敲除单个TspanC 8，并使用转录荧光素酶报告基因测定来测量Notch 1活性。在Jurkat细胞中的发现将扩展到具有不同类型的Notch 1激活突变的T-ALL细胞，例如KOPTK 1和P12 ICHIKAWA细胞。B）为了研究Notch 1在内化后是在细胞表面还是在囊泡上被切割，我们将使用共聚焦和结构化照明成像，以及荧光相关光谱，对转染的荧光标记的TspanC 8进行成像，C）为了确定靶向Notch 1的cDNA是否抑制T-ALL增殖我们已经显示在Jurkat细胞中敲除ADAM 10后增殖减少（图1B），与公开的数据一致14。此外，Tspan 14敲低和Tspan 5抗体的组合抑制配体诱导的Notch 1信号传导9。我们将测试TspanC 8敲除或TspanC 8抗体治疗是否会抑制T-ALL细胞系和原代细胞的增殖。我们最近产生了Tspan 14和Tspan 15单克隆抗体（Tomlinson实验室，未发表）。我们将与业界合作生产更多的抗体; 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 We have recently generated Tspan14 and Tspan15 monoclonal antibodies (Tomlinson lab, unpublished).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.