Ubiquitin-dependent trafficking and turnover of the immune checkpoint protein CTLA4.

Most of the time our bodies' immune system operate with the brake on. This is crucial for self-tolerance and stops our cells being attacked indiscriminately. However, cancer cells can generate novel molecules which are recognised by our immune system. Revving up the immune response in this context can be highly beneficial and this concept underpins what is now known as immunotherapy. Here, the immune system is let off the leash to fight the cancer, by removing the normal braking system. The therapies that have been developed are antibodies which recognise cell surface receptors and block their interactions. One of these receptors, called CTLA4, has a fleeting existence; after being made in the cell it will be gone within the hour. It is consigned to the cell's "rubbish dump" or "lysosome", to be recycled for parts. Short-lived proteins, such as CTLA4, are normally recognised by other proteins which label them with a degradation tag or "signal" called ubiquitin. The identity of the protein that adds a ubiquitin tag onto CTLA4 and condemns it to be destroyed is unknown. We plan to discover it by sifting through about 600 candidates in a highly parallel fashion. Alongside identifying this key molecule, we will also seek to understand any nuances of the CTLA4-ubiquitin signal and identify the molecules that recognise it and usher CTLA4 to the lysosome. By understanding this basic cellular machinery that controls cellular CTLA4 levels we aim to open up new strategies for their adjustment in a therapeutically meaningful way.

The immunotherapy revolution is ongoing, but the underlying understanding of the dynamics of immune check-point receptors is relatively under-developed. Amongst these, CTLA4 stands out as a very short-lived protein, which implies that its rate of degradation provides a key control mechanism that determines cellular levels.

We have recently determined that CTLA4 ubiquitylation dictates its lysosomal degradation, but the relevant E3-ligase is not currently known. We will seek this key regulator using CRISPR/Cas9 screening with a bespoke pooled library of gRNAs targeting ~600 ubiquitin E3 ligases. Importantly, this short half-life is conserved across cell types, which allows us to engineer cell models for our screen that express a tagged version of CTLA4. With these in hand, we will implement a FACS screening approach that will allow us to separate cells with high CTLA4 from those with low CTLA4 surface levels. We will quantitate the representation of gRNAs within these cell populations to identify candidate E3s which will then be characterised in a more intricate manner. We thereby hope to identify the critical conveyor of the ubiquitin signal, whilst in other work packages we will explore the ubiquitin chain topology and identify those molecules whose interaction with CTLA4 is contingent on ubiquitylation.

This combination of work packages will generate a molecular understanding of the key means by which CTLA4 levels are controlled and open up therapeutic opportunities for small organic molecule drug development, that might complement the existing biological agents.