Understanding and targeting oncogenic biomolecular condensates of ALK kinase

Lung cancer is the 3rd most common cancer in the UK, with almost 50,000 new cases diagnosed annually. Lung cancer is classified as small cell or the more common non-small cell (80% of cases). Approximately 5% of these patients have a specific mutation in the DNA of their cancer cells, resulting in a gene fusion called EML4-ALK. EML4-ALK patients are on average much younger than most lung cancer patients. They are treated with ALK tyrosine kinase inhibitors, and 5-year survival for these patients has improved to 50%-60%, much more effective than standard chemotherapy. There is still room for improvement, and so we need to decipher the molecular mechanisms through which EML4-ALK proteins promote cancer cell survival and proliferation and to determine how the cancers become resistant to ALK inhibitor therapy. This will allow us to develop new and optimised treatments.

In this project, we will focus on the molecular interactions of EML4-ALK proteins, the cellular structures they form, and how these respond to cancer therapies. In healthy cells, EML4 regulates the formation of cellular structures called microtubules. The ALK protein normally functions on the surface of cells in the developing brain where, in response to a signalling molecule from outside the cell, it instructs the cell to proliferate. In lung cancer, parts of these proteins are fused together, and interactions between EML4-ALK protein molecules allows them to instruct lung cancer cells to proliferate out of the control of the rest of the body. These interactions also allow EML4-ALK proteins to form compartments within cells that are enriched in other proteins required to promote cell proliferation and survival. We previously discovered that some ALK inhibitors used in cancer treatment destabilise the compartments, whereas one commonly used ALK inhibitor stabilises the compartments. Based on this and other observations, we think that the compartments are formed from interactions between the ALK parts of the EML4-ALK protein molecules, as well as interactions between the EML4 parts. If we could block these interactions, this might lead to a new way to treat EML4-ALK lung cancers. We will focus on the ALK interactions because we think this is more feasible, and because uncontrolled ALK activity is the cause of many other cancers, whereas EML4 dysfunction is rarely found in other cancer types.

Our project will begin with the production of molecular tools to disrupt the ALK interactions. We have already made a set of eight synthetic protein molecules called nanobodies that bind to EML4-ALK in cells. We will develop these molecular tools further by characterising precisely how they affect ALK interactions and activity. In parallel, we will determine precisely how ALK molecules interact with each other. Both strands of work will use experimental structural biology methods and computational methods such as artificial intelligence. Also in parallel, we will find out which other proteins interact with EML4-ALK and determine which of them are present in the cellular compartments. The organisation of these proteins within the compartments, and the movement of molecules in and out of the compartments will be studied using state-of-the-art cell imaging methods. This part of the project will tell us how the compartments are built, and how molecules are passed from the compartment to the rest of the cell with the instructions to proliferate.

The final step of the project will be to use this new information and our new molecular tools to ask whether disrupting or stabilising the compartments can effectively block the cell proliferation and survival signals from EML4-ALK. We will test this in cells that were cultured from lung cancer patients, and cells that have become resistant to ALK inhibitors used in cancer therapy . If successful, we will initiate a follow-up project towards improving the treatment of lung cancer based on our findings.

EML4-ALK gene fusion is the key oncogenic driver event in ~5% of non-small cell lung cancer (NSCLC) patients. EML4-ALK variants generate proteins with different biological properties. EML4-ALK V3 is a marker of poor response to ALK inhibitor therapy and more aggressive, metastatic disease. EML4-ALK V3 protein forms dynamic, liquid-like compartments in the cytoplasm of cells, whereas EML4-ALK V1 compartments are static and solid. Other signalling molecules such as GRB2 are recruited to the compartments, and ALK inhibitors can either disrupt or stabilise them.
We hypothesise that the cellular signalling compartments are built on a scaffold of EML4-ALK proteins, formed by interactions between the EML4 trimerisation domains (TD) and between ALK kinase domains in their active conformations. Additional proteins propagate signalling and may also contribute to the structure of the compartment.
This project aims to unravel the molecular basis of key interactions that drive EML4-ALK compartment formation and function, and determine how manipulation of these interactions affects compartment structure and signalling, through three workpackages:
(WP1) characterise ALK self-association using computational and biophysical approaches, including X-ray crystallography and structural mass spectrometry (WP1); disrupt or stabilise ALK-ALK interactions using bespoke nanobodies;
(WP2) resolve how protein-protein interactions contribute to compartment structure and function using BioID to identify EML4-ALK binders; cell imaging and knockdown approaches to validate compartment proteins; and super-resolution imaging to reveal the organisation and dynamics of compartment proteins;
(WP3) disrupt oncogenic signalling in lung cancer cell lines that are resistant to ALK inhibitors using nanobodies and structure-based mutations. This will address whether disruption or stabilisation of compartments is a preferred strategy to block EML4-ALK signalling in ALKi-sensitive or resistant cell-lines