The charity is currently funding two important pieces of research in the UK, and one project to develop a UK national EHE biobank. We believe the tow research projects 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 explanation of the gene mutation and resultant fusion protein that are believed to drive EHE development, and their interaction with a critical 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 and found in 90% of EHE tumours) is causing tumour development. 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 such models are incredibly limited at the current time, 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 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 very limited availability of both an EHE cell line and EHE animal models 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 currently being tested.
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.
Following her introduction to the Charity by Dr Rubin, 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 (pathways) 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. As explained above, the dysregulation of the Hippo Pathway is believed to be a key component of EHE biology.
The PhD project, which started in January 2019, aims to determine how the TAZ-CAMTA1 fusion protein, frequently found in EHE tumours, affects the biological characteristics of endothelium, the cell type affected in EHE. It is the aim of the project to define how the expression of this aberrant fusion protein alters endothelial cell identity and behaviour such as proliferation, migration or interaction with other cells. It is hoped that addressing these questions will provide a better understanding of the cellular and molecular mechanisms underlying this rare vascular cancer.
To address this series of questions, the project is taking advantage of a model system established in the Kouskoff laboratory. In this experimental system, embryonic stem cells are differentiated to form endothelial cells in which the expression of TAZ-CAMTA1 can be induced by the addition of doxycycline.
To date, a series of experiments have been performed to evaluate culture conditions for the generation of endothelial cells from differentiating embryonic stem cells. Using selected optimal conditions, the project has shown that expression of the TAZ-CAMTA1 fusion protein at the early stage of endothelial formation has a strong negative impact on this cell type. It was observed that upon TAZ-CAMTA1 induction, the expression of key endothelial proteins was significantly decreased. Preliminary experiments using a TAZ-CAMTA1 mutant protein that cannot interact with critical molecular partners showed that the mutant fusion protein did not alter the expression of these key endothelial proteins. Further work is currently ongoing to determine how TAZ-CAMTA1 expression produces its effect, whether it is through changes in proliferation, control of gene expression or induction of cellular death.
Dr Valerie Kouskoff also hopes the project will be able to create induced pluripotent stem cells using patient-donated tumour cells, when available. These pluripotent stem cells will represent a unique and powerful approach to study the biology of EHE. The research will aim to understand better how the EHE cancer cells arise and proliferate. This research will also aim to generate and maintain enough EHE cancer cells in the laboratory to test drugs that could stop the proliferation or kill these cells. Carrying out this part of the research however requires tissue samples from patients with EHE, and this remains a key focus for the charity moving forward.
Project: Development of a UK EHE tissue management and biobanking capability.
(The Joint Royal Marsden-ICR Sarcoma Research Unit, ICR, London)
One of the key areas that may hinder wider research into EHE is the lack of appropriate patient samples, such as blood and tissue, with which to work. The charity was in discussion with two U.K. institutions in 2016/17 with regard to setting up an EHE biobank to ensure that such samples are captured going forward, but failed to progress these discussions with either group. However, in early 2019 the charity reached agreement with the Royal Marsden Cancer Charity to fund a new Tissue Manager position at the Institute of Cancer Research, within The Joint Royal Marsden-ICR Sarcoma Research Unit. Through this agreement we plan to establish a UK-wide coordinated EHE biobanking capability. This project is being lead by Dr Paul Huang at the ICR.
The role started in June 2019, and initially focused on collating and organising the historic EHE sample and clinical record data set held by the Royal Marsden Hospital. Shortly thereafter, work started on the documentation and organisation of the biobank itself. At the end of March 2020, the Tissue Manager had prepared all procedures and documents for the biobank. The Royal Marsden Hospital have also been very helpful, agreeing that the actual biobank sampling and storage procedures will be undertaken by the RMH Generic Tissue Bank (GTB), who already run and administer biobanks for several other cancers.
The team are now ready to move forward with the ethics approval which will be the last stage of the set up procedures before the biobank goes live. After this the EHERCC and the ICR will be working together to publicise the biobank and encourage participation of all EHE patients and hospitals in the UK. This is exciting. Tissue and fluid collection is important to assist with EHE research. Up until now it has been an adhoc process with individual patients and the charity working with hospitals on a case by case basis to try and save samples for future research. With this fully-approved EHE national biobank open to the whole UK, and with an accepted sampling programme for each patient, the Charity hopes that tissue and fluid sampling and capture will become coordinated, planned and very effective.
The EHERCC is now working with the biobank team to assist with the completion of the biobank set-up and the subsequent publicity of its launch. Initial focus is currently on the UK national process and getting this up and running.