The charity is currently funding two important pieces of research in the UK. We believe these are the first two EHE-dedicated research projects to be undertaken in the United Kingdom. The first project, involving the development of an EHE zebrafish model, is based at the University of Sheffield. The second, a PhD project looking at how EHE impacts endothelial cells, is based at the University of Manchester. Summaries of both projects are provided below. To assist with understanding these summaries, we have first provided a high-level explantion of the gene mutation and resultant fusion protein that are believed to drive EHE development, and their interaction with a critcal process in our cells known as the Hippo Pathway.
TAZ-CAMTA1 and the Hippo Pathway
Our first two EHE research projects in the UK are both focused on the biology of EHE, and in particular how the EHE disease-defining WWTR1-CAMTA1 gene mutation (discovered by Dr Rubin in 2011) is causing tumours. Current understanding strongly suggests that this gene mutation results in the generation of a new protein in our cells, referred to as a fusion protein. Dr Rubin was able to identify and refine this fusion protein in 2017, and has shown that it is created by the joining of two existing proteins in our body, TAZ and CAMTA1. It appears that the new TAZ-CAMTA1 fusion protein is then deregulating, or interrupting, the Hippo Pathway, a fundamental biological process in our cells that controls cell division and proliferation. In EHE this process is occurring in endothelial cells and results in the formation of tumours in the lining of blood vessels.
So clearly understanding the impact that TAZ-CAMTA1 has on our cells is very important. Equally important is research that can help identify compounds and drugs that may ultimately block the fusion protein. Developing in vitro and in vivo models of EHE is also critical, as no such models currently exist, but are vitally important for testing hypotheses about EHE and for ultimately testing possible now drug regimens. It is these critical research areas that our two current research projects, one at the University of Sheffield and one at the University of Manchester, are both focused on.
Project: Using Zebrafish to study TAZ-CAMTA1
(Bateson Centre, University of Sheffield):
In 2018 EHERCC provided funding for a new project led by Dr Fredericus Van Eeden at the Bateson Centre at the University of Sheffield to use zebrafish to create a pre-clinical model of EHE. Dr Van Eeden is a research scientist with expertise in this field and Trustees were very pleased to visit his laboratory in Sheffield in February 2019 and meet him and his staff to discuss their work. This project compliments the research from Dr Brian Rubin’s laboratory at the Cleveland Clinic in Ohio, also supported with funds from EHERCC, and in fact was assisted in the early stages by Dr Rubin’s laboratory providing the necessary EHE clones of the human WWTR1-CAMTA1 gene mutation.
It is recognized that EHE is caused by the WWTR1-CAMTA1 genetic mutation that creates the TAZ-CAMTA1 fusion protein in EHE. However, a key challenge of understanding EHE as a disease is the absence of both an EHE cell line and an EHE animal model to use for research purposes. The aim of this research project is therefore to primarily create an EHE zebrafish model by introducing the gene mutation that will then express the TAZ-CAMTA1 fusion protein, and then use this model to observe what happens when the fusion protein is expressed in zebrafish.
The zebrafish has become an important vertebrate model organism in scientific research and more recently for studying human disease. It shares 70% of its genome with humans, it is small and robust, inexpensive to raise, easy to breed and as the zebrafish embryos are transparent their blood vessels can be seen easily using a low power microscope. This last feature is of great importance as it will enable changes in the development of the zebrafish to be observed after the fusion protein has been produced.
The project aims to develop a zebrafish model that expresses the EHE mutation that produces the fusion protein and then to describe any changes to the morphology of the blood vessels as the zebrafish develop. This will help in the understanding of EHE. In addition, if a genetically modified zebrafish can be created, it will provide a low-cost model to use for the testing of drugs that might be beneficial for treating EHE.
Initial results were very encouraging with the WWTR1-CAMTA1 genetic mutation successfully introduced into the zebrafish. However, on further detailed evaluation, it was noted that the gene mutation was not present in endothelial cells. This surprising result required the research team to explore and develop alternative methods to introduce the gene mutation with the hope of seeing the mutation in transgenic fish. Three alternatives have been developed and are being testing at the mid-point of 2019. Preliminary indications are that at least one of these alternatives may have worked.
Project: An in vitro model system to study the molecular and cellular features of EHE
(Department of Developmental Biology and Medicine, University of Manchester)
The tumours found in EHE originate in the endothelial cells that line blood vessels, and are mainly found in the inside lining of blood vessels, lymph vessels, and the heart. But we don’t understand what happens in these cells to cause the tumours to develop. A better understanding of the disease biology and the resultant effects on endothelial cells will be crucial to developing therapies.
EHERCC is funding a PhD project led by Dr Valerie Kouskoff at the Department of Developmental Biology and Medicine at the University of Manchester using embryonic stem cells as an in vitro model system to study the effects of the TAZ-CAMTA1 fusion protein. Dr Kouskoff’s group has extensive experience in the genetic manipulation of embryonic stem cells, and in studying the signalling mechanisms (pathway) that allow them to develop into endothelial cells. She is also a recognised expert on the Hippo Pathway, one of the most fundamental pathways in cells that is key to the regulation of cell development and proliferation.
In this project, a WWTR1-CAMTA1 gene that has been genetically engineered to be controlled by a molecular ‘switch’ will be introduced into embryonic stem cells. This will allow the researchers to record what happens when the TAZ-CAMTA1 fusion protein is ‘switched on’ in the cells, and to better understand how TAZ-CAMTA1 might interfere with the behaviour and identity of endothelial cells in EHE.
In addition, it would be extremely valuable to establish in vivo models of EHE. Dr Kouskoff’s group will engraft endothelial cells that express the TAZ-CAMTA1 fusion protein into mice. If successful, this mouse model may allow researchers to assess under what conditions EHE tumours spread around the body, assess what happens when the disease progresses from the indolent to aggressive phase, and test potential therapies.
Significantly, in normal cells, the TAZ part of the fusion protein is involved in regulation of the Hippo Pathway. The Hippo pathway is known to be implicated in several more common cancers. Therefore, a better understanding of the mechanism that leads to EHE, and the involvement of TAZ in this, is likely to provide insights into other cancers as well as EHE.