Understanding how tetraspanins and the ‘molecular scissor’ ADAM10 promote blood cancer

Steve Briddon (University of Nottingham) 3rd supervisor. Nick Holliday (University of Nottingham) 4th supervisor.

Introduction
T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive blood cancer that is in urgent need of more effective therapies. T-ALL carries a lifetime risk of less than 1 in 2000 people, but risk increases with age and standard chemotherapy is ineffective in 40% of patients. Over 65% of T-ALL is driven by activating mutations in the cell fate regulator Notch1, which allow its activation by the ‘molecular scissor’ ADAM10. The cleavage releases the Notch1 intracellular domain to act as a transcription factor to induce malignant proliferation. However, therapeutic targeting of ADAM10 is not viable because of toxicity; ADAM10 is expressed on all cells and has numerous substrates that are important in health.

We have discovered that ADAM10 substrate specificity is dictated by its association with one of six regulatory tetraspanins, termed TspanC8s - Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33. We propose that ADAM10 is not one scissor, but six different scissors with different substrate preferences, depending on the associated tetraspanin. The tetraspanins are a superfamily of 33 transmembrane proteins which regulate the localisation and clustering of interacting partners like ADAM10. The first structural characterisation of a full-length tetraspanin, published by the Blacklow group in the journal Cell in 2016, suggests that they regulate partner protein function by conformational change (i.e. as ‘molecular switches’), and thus have great potential as therapeutic targets. Targeting specific TspanC8-ADAM10 complexes might modulate specific disease-associated substrates, without the toxic side effects that would be associated with global ADAM10 targeting.

Hypothesis
A specific TspanC8/ADAM10 scissor drives T-cell acute lymphoblastic leukaemia (T-ALL) by specifically cleaving mutant Notch1, and is thus a promising therapeutic target for T-ALL treatment.

Experimental methods and research plan
1) To determine which tetraspanin/ADAM10 scissor(s) activates Notch1 in T-ALL
Individual TspanC8s will be knocked out using CRISPR/Cas9 in cultured T-ALL cell lines. Knockouts will be validated by western blotting using the Odyssey Infrared Imaging System and by flow cytometry. Notch1 activity will be measured using a transcriptional luciferase reporter assay and by quantitative PCR to detect Notch1-induced genes.
2) To investigate whether mutant Notch1 is cut at the cell surface or on vesicles following internalisation
Advanced fluorescent microscopy will be used to image fluorescent-tagged TspanC8s, ADAM10 and a Notch1 cleavage-sensor in T-ALL cell lines, to determine where mutant Notch1 activation occurs. Imaging techniques will include confocal, total internal reflection fluorescence (TIRF), fluorescence correlation spectroscopy (FCS), lattice light-sheet microscopy, and super-resolution techniques such as structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM).
3) To determine whether targeting the scissor for mutant Notch1 inhibits T-ALL proliferation
Knockout of TspanC8s, or treatment with TspanC8 antibodies, will be assessed for anti-proliferative effects on cultured T-ALL cell lines and primary cells from patients.

Expected outcomes and impact
The experiments will determine which of the six TspanC8/ADAM10 scissors activates mutant Notch1 in T-ALL, where in the cell this activation occurs, and will assess TspanC8 antibodies for future T-ALL therapy.

Person Specification
Applicants should have a strong background in cell biology, and ideally a background in molecular biology. They should have a commitment to research in cancer biology and hold or realistically expect to obtain at least an Upper Second Class Honours Degree in a subject within the biosciences.