In this section we provide a high-level summary of the progress that has been made in the research we are either already funding or are in discussions to start. As research by its nature may take time to see material progress we will update this section of the website on a six monthly basis. We will of course also update with specific significant events as appropriate.
Dr Rubin’s research
The following section details progress in the research being led by Dr Brian Rubin at the Cleveland Clinic in Ohio, USA.
A. Genetic basis of EHE tumor progression
Dr Rubin hypothesized that disease progression in EHE is due at least in part to the accumulation of genetic mutations that interact with the WWTR1-CAMTA1 gene fusion that is found in virtually all EHE’s. This work has now been completed and was presented at the annual meeting at the Connective Tissue Oncology Society in November, 2017. Dr Rubin is now in the process of writing the manuscript which describes how more aggressive EHE’s do in fact possess additional mutations such as loss of CDKN2A in addition to the canonical WWTR1-CAMTA1 gene fusion, which is found in all cases of EHE.
B. Therapeutic compounds that target TAZ-CAMTA1
TAZ-CAMTA1 is the protein encoded by the WWTR1-CAMTA1 gene fusion. Dr Rubin believes that TAZ-CAMTA1 is the central oncogenic driver that underlies all EHE biology. As a corollary, Dr Rubin believes that if we can inhibit TAZ-CAMTA1 then EHE can be controlled. Dr Rubin’s team spent the last year optimizing expression of proteins and protein fragments that are used to construct the assay. They discovered that they could produce good quantities of all proteins using E. coli expression systems and codon optimization. After establishing how to produce the proteins, Dr. Che, who leads this research strand, worked on purifying these proteins to high purity, which she also achieved. Finally, she was able to put the proteins together in an Alphalisa-based assay (Perkins Elmer proprietary technology) and she has performed extensive characterization of this assay. At the end of 2017, Dr Rubin’s team are in the process of moving the assay into the Case Western Reserve Screening Core to screen a 50,000 molecule Chembridge small molecule library for the ability to inhibit the interaction between TAZ-CAMTA1 and TEAD4. This interaction is critical for TAZ-CAMTA1 function and an Achilles’ heel that Dr Rubin’s believe’s can be exploited. After the initial screen which will take place in Q1 of 2018 Dr. Che will perform secondary, tertiary, and quarternary screens to find the best candidates for further drug development. By the end of the year Dr Rubin hopes to have identified at least one chemical series to move into drug development.
C. Genetically Engineered Mouse (GEM) EHE model
Dr Rubin’s team have been working on a genetically engineered mouse model of EHE and at the end of 2017 the model is ready for testing. The construction phase of this project began in 2015 and has been ongoing until July 2017. The team had hoped to have the mice earlier but encountered setbacks targeting their targeting constructs in mouse stem cell lines. The EHE model contains a Wwtr1-Camta1 gene fusion targeted to one endogenous Wwtr1 allele but the fusion is in an inverted orientation “off” and flanked by loxP sites such that expression of Cre-recombinase results in a recombination event that “turns on” the mutant fusion. If the fusion gene can be activated in the appropriate cell type, Dr Rubin hopes that EHE should be seen. The beauty of this model is that they can turn the fusion gene on when and where they want. The team have turned on this allele in the germline and it is an embryonic lethal. Not surprisingly, the embryos do not survive. They are currently trying to understand why the embryos die. They are also turning the gene on in adult mice using two approaches. The first approach is to turn it on in the endothelial lineage using an “Endoglin/CD105 Cre” that was developed by Ralf Adams at the Max Planck Institute and obtained from Taconic Biosciences, who licenses the mouse. They have also developed a mouse that turns the fusion gene on in all lineages using a Rosa Cre mouse developed by Tyler Jacks at MIT. Both Cre recombinases are induced/stabilized by tamoxifen so the team inject young mice with tamoxifen and wait to see if they develop EHE. So far they have analyzed 4 young endoglin Cre/fusion gene mice and they were negative for lesions but the team have many more mice to analyze. By the end of 2018 Dr Rubin will know whether either of these approaches has worked to generate a genetically engineered EHE mouse model. This experiment is high risk but also high reward as currently there is no bona fide EHE mouse model.
D. Proteomics analysis of TAZ-CAMTA1 binding proteins
The proteins that bind to the TAZ portion of TAZ-CAMTA1 are relatively well documented since TAZ has been studied broadly. However, much less is known about proteins that bind to CAMTA1. Dr Rubin is interested in proteins that bind to CAMTA1 since some of these proteins may be critical to the function of TAZ-CAMTA1. To determine proteins that bind to the CAMTA1 portion of TAZ-CAMTA1, his team have removed portions of CAMTA1 from the protein using targeted genetic approaches. They are able to express these proteins and using protein “tricks” they are able to purify the different versions of TAZ-CAMTA1 along with the proteins that bind to them. They then use mass spectrometry to identify the proteins that bind to TAZ- CAMTA1. Using this approach, his team have identified several interesting candidate proteins that bind to the CAMTA1 portion of the protein. Dr Rubin will examine their function Q1 and Q2 this year to determine if inhibition of these protein interactions has functional significance. These experiments may reveal additional therapeutic targets.
E. Trametinib clinical trial
Based on preclinical data developed in Dr Rubin’s laboratory and presented at the CTOS annual meeting in 2017, Dr Rubin’s team have developed a clinical trial in conjunction with SARC to examine the efficacy of trametinib, a MEK/MAP kinase pathway inhibitor.
This is the first prospective clinical trial in EHE and the Principal Investigator is Dr. Scott Schuetze from the University of Michigan. The trial opened in May 2017 and accrual to the first phase of the study is already complete at 14 patients. If there is a single objective response in the first phase, a second phase will open and 13 additional patients will be accrued. Dr Rubin’s laboratory will perform correlative studies to document WWTR1-CAMTA1 gene fusions in patients and to examine core biopsies to determine whether trametinib suppresses MEK in the patients EHE tumors. Also, if patients respond and then progress we will study the mechanism of resistance in these patients. At the end of this trial we should be able to answer the question of whether trametinib is useful in the treatment of progressive EHE.
UK EHE research and initiatives
Dr Rubin’s research is not the only research currently being discussed with different researchers. The following is a brief summary of EHE research that the charity is funding in the UK .
PhD under Dr Valerie Kouskoff
Dr Valerie Kouskoff is leading a University of Manchester research team in a new study of EHE, which was initiated following collaboration with Dr Rubin. This research will use new methods not previously used to study EHE.
The purpose of this project is to create stem cells that have all the molecular characteristics of EHE tumor cells and that will allow studying EHE in a novel biological context. Powerful stem cells can be created (or “induced”) by changing cells from your body, such as skin cells or cancer cells, into pluripotent stem cells in the laboratory. “Pluripotent” means that the cells can turn into any kind of cells such as endothelium, blood, brain, heart, or kidney cells. For this reason, induced pluripotent stem cells can be used to study, and maybe one day help treat, diseases or injuries that have caused patients’ cells to die or become damaged.
Dr Valerie Kouskoff hopes to create induced pluripotent stem cells using patient-donated tumour cells. 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 research requires tissue samples from patients with EHE, and this remains a key focus for the charity moving forward. However, Dr Kouskoff has not been idle while awaiting rare tissue donations. She has created embryonic stem cells carrying the EHE fusion protein with the ability to turn the expression of the protein on and off at different points during endothelial differentiation, to see what effects this may have on these cells, and so hopefully build a better understanding of how the TAZ-CAMTA1 fusion protein can influence endothelial development and provide a unique window that may provide insight into how EHE develops.
The Hippo Pathway is a key process in the body that regulates the generation and growth of new cells as well as the death of old cells, and ultimately controls the size of all human organs. A key component of EHE is the dysregulation of the Hippo Pathway, leading to the proliferation of EHE cells and resultant EHE tumours. Dr Kouskoff has identified an important role for the Hippo pathway in blood and endothelial development so this work in EHE is a natural extension of her prior studies.
We were delighted to conclude all discussions with Dr Kouskoff and the University of Manchester in late 2018 and to commit to funding her proposed work, as a PhD project, in her lab. Further details about Dr Kouskoff's research are included in the next section of this website, "UK EHE research".
Zebra Fish model
In addition to Dr Rubin’s mouse model, the EHERCC also investigated the setting up of a collaboration between an institute in the U.K. and Dr Rubin to explore the feasibility of creating a valid EHE model using zebrafish. These fish are used widely to model human diseases due to a large degree of genetic similarity. The attraction of zebrafish is that they breed and grow very quickly and in large numbers. A valid model therefore allows for greater and faster testing and experimentation of drugs and hypothesis-driven research on a living organism.
Some very preliminary experimentation had previously shown some interesting results, and we hoped we could build on this idea. As a result, the Charity entered into discussions with Dr Fredericus Van Eeden at the Bateson Centre at the University of Sheffield, through Dr Robin Young who is also based at the University of Sheffield, with a view to agreeing a suitable project. We were extremely pleased to conclude all discussions with Dr Van Eeden in the first half of 2018, with commitment to funding a one-year programme to develop the zebrafish model. The work started in August 2018, and has been extended for a second year. Further details about the zebrafish project are included in the next section of this website, "UK EHE research".
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 with regard to setting up an EHE biobank to ensure that such samples are captured going forward, but failed to reach agreement 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 hope to eventually establish a UK-wide coordinated EHE biobanking process. The role started in mid June 2019.
Other EHE research
The EHE Foundation in the USA is in advanced discussions with one of America’s pre-eminent specialists in the field of immunotherapy, with a view to undertaking research to explore the development of ‘personalised vaccines’ for EHE patients. This project will start with the premise that fusion proteins involved with a cancer can act as an antigen which will be recognised by a patient’s immune system and attacked. We know that the TAZ-CAMTA1 fusion protein is a key driver in EHE. So the first stage of the project will examine whether this fusion protein is indeed recognised by our immune cells.
In addition to the TAZ-CAMTA1 fusion protein, the project will also genetically sequence samples from approximately 40 patients, comprising 20 progressive and 20 indolent cases. This sequencing will be used to identify other gene fusions and then predict likely antigens that will be specific to that patient’s tumours. Once these groups of neoantigens have been identified, they can be used as a vaccine and re-introduced into a patient to stimulate the patient’s own immune system, educating their T cells to attack the cancer in their bodies. In some cases the use of the vaccine may be enhanced with the use of different immunotherapy drugs.
This project represents an exciting possibility for combatting EHE. Encouragement for the research is provided by the fact that EHE is seen to go into remission in a very few patients, suggesting that in these cases, the patient’s own body has begun to recognise and attack the cancer cells. In addition, in some cases, female patients have seen their EHE tumours disappear during pregnancy, again suggesting a strong response from their immune system, possibly linked to the strengthening of the female immune system in the last trimester of pregnancy.
Biomarkers are “things” found in the blood that can be attributed to a particular type of cancer. These biomarkers may be small parts of tumour DNA, or specific proteins or other components of the cancer that are released into the blood when tumour cells die.
Biomarkers once identified are important as they may act as an early warning sign that a patient has cancer, or that the cancer is changing, perhaps becoming more aggressive. In both situations early warning may allow doctors to act more quickly and initiate a new drug regimen earlier, and so increase the chance of achieving a positive result.
The EHE Foundation in the USA is in late stage discussions with doctors and researchers at one of America’s pre-eminent medical clinics regarding adding EHE into their ongoing research to identify biomarkers.