Programme details

 

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 either on a six monthly basis or with specific significant events when they occur.

 

Dr Rubin’s research

The following section details progress in the research being led by Dr Brian Rubin MD, PhD 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 secondary genetic mutations that interact with the WWTR1-CAMTA1 gene fusion that is found in virtually all EHE’s. This work was completed and was presented at the annual meeting at the Connective Tissue Oncology Society (CTOS) 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.

 

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, and therefore, developing an assay to allow the screening of drugs that might inhibit or prevent the effects of this fusion protein is a key focus.

Dr Rubin’s team spent most of 2017 and early 2018 optimizing expression of proteins and protein fragments that could be used to construct such an 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 the team worked on purifying these proteins to high purity, which they achieved.

Finally, they were able to put the proteins together in an Alphalisa-based assay (Perkins Elmer proprietary technology) and complete extensive characterization of this assay. Dr Rubin’s team then moved 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 believes can be exploited.

The initial screen which took place in mid- 2018 produced a number of very interesting results. The team then completed secondary, tertiary, and quarternary screens through 2018 and into 2019, to find the best candidates for further drug development. Work in progressing the identification and development of these possibly-therapeutic compounds will not be quick, but the work is progressing. We hope to be able to provide additional more detailed information in the near future.

The Charity is particularly pleased that Dr Che, who has been instrumental in leading the lab work, has played such a key role in these exciting developments, as funding for Dr Che’s position was the first grant provided by the Charity in early 2016. The charity, together with its sister EHE foundations, also funded the Perkins Elmer proprietary technology and the use of the Case Western Reserve Screening Core, both of which have been core to Dr Rubin’s important compound-screening research.

 

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 was ready for testing. The construction phase of this project began in 2015 and progressed until the end of 2017. The team had hoped to have the mice earlier but encountered setbacks targeting their 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 believed that EHE should be seen.

The beauty of this model is that the fusion gene can be turned on when and where researchers want. The team have turned on this allele in the germline and it is an embryonic lethal. Not surprisingly, the embryos do not survive. Work therefore in 2018 was focused on trying to understand why the embryos die. They are also turning the gene on in adult mice using two approaches. Initially the mice continued to fail to present with EHE tumours. This experiment was high risk but also high reward as there was no bona fide mouse model for EHE. Dr Rubin’s team, in collaboration with other mouse model experts have continued to work with the mice through 2019 to identify why tumours are not developing.

Then in late October 2019, Dr Rubin was able to announce the development of the world’s first EHE Mouse Model. Dr Rubin was delighted with the progress achieved and the apparent quality of the model. “I've done a thorough review of the mice that have been used to generate histology so far and we are seeing EHE's in all anatomical areas seen in humans. Additionally, I have immunohistochemical verification (CD31 and CD34 positivity) that the lesions do show endothelial differentiation. This model is remarkable and truly does represent a breakthrough. We are now working on freezing tissue for development of cell lines, xenografts, and for molecular/expression profiling analysis. We will spend the next several months developing cell lines from these tumors as well as characterizing the tumors at the molecular (RNA and DNA) level. This is the first bona fide biological model of EHE so it will open up a lot of potential studies for us and our collaborators.”

We congratulate DR Rubin on this impressive break-through in such a key area. We will of course be updating this section as we see further progress with these EHE mice.

 

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 is examining their function 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 was completed at 14 patients. The first phase of the trial generated the identified objective response in one patient and as a result the second phase of the trial was initiated and is ongoing, with patients still being recruited. Dr Rubin’s laboratory are performing correlative studies to document WWTR1-CAMTA1  gene fusions in patients and to examine core biopsies where available to determine whether trametinib suppresses MEK in the patients EHE tumors. Also, if patients respond and then progress, his lab will study the mechanism of resistance in these patients. At the end of this trial the trial team hope to be able to answer the question of whether trametinib is useful in the treatment of progressive EHE.

 

Other EHE research and initiatives

Dr Rubin’s research is not the only research now being funded by the Charity which continues to support different researchers. The following is a brief summary of additional EHE research that the charity is funding outside the UK. In addition, the Charity is now funding EHE research projects in the UK. Information about these UK projects can be found on the UK EHE RESEARCH page that follows this page within this Research section of our website.

 

Dr John Lamar

In late 2018, the Charity was approached by Dr Guy Weinberg with a view to funding a new piece of research. Dr Weinberg, founder of the Cravat Foundation, had in 2016 also established his Telluride YAP, TAZ & TEAD workshop, which is held each year in Colorado. This workshop was started in order to bring clinicians and researchers together who were working with YAP and TAZ, two important proteins that are involved in the biology of many cancers. EHE is of interest to such researchers as both YAP and TAZ are believed to be part of the biology of EHE, possibly deregulating the Hippo Pathway, a fundamental cellular process in our bodies that controls cell growth and proliferation. Because EHE is believed to be a very simple cancer it is therefore possible that EHE may provide a useful model for researching exactly how YAP and TAZ act in the development of cancer. This could have significant implications not only for EHE but for other forms of cancer, including some of the common forms of the disease.

The grant application, supported by Dr Rubin, was submitted by Dr John Lamar, based at the Albany Medical College in New York. The research is looking to test the hypothesis that the TAZ-CAMTA1 fusion protein that is core to EHE is subject to regulation by Hippo-pathway independent mechanisms that could be exploited to inhibit TAZ-CAMTA1 function and so treat EHE. Dr Lamar intends to use his extensive expertise in developing mouse xenograft models to assay cancer growth and progression in vivo (see objective 2 below).

The project has two core objectives:

  1. To identify regulators of the TAZ-CAMTA1 fusion protein and assess their therapeutic potential; and
  1. To establish model systems to assay TAZ-CAMTA1 function in vitro and in vivo and use to test therapeutic potential of candidate TAZ-CAMTA1 regulators.

 

The EHE foundations collectively agreed in mid-2019 to sponsor this interesting new research proposal which will be evaluating different therapeutic possibilities.

The project team have already been able to establish and confirm TAZ-CAMTA1 expression in mouse cells. In these cells they can also demonstrate significantly higher transcription involving YAP/TAZ-TEAD when compared to control cells and have shown that this is due to the TAZ-CAMTA1 fusion protein.

The next step was to test a number of possible genes to see if they could repress the TAZ-CAMTA1 fusion protein. The team found that 6 of the genes tested significantly repressed TAZ-CAMTA1 activity and that this repression was consistent across several experiments. These 6 proteins each has a role in existing cellular pathways, so if the research team could find a way to activate these pathways in EHE cells it could inhibit TAZ-CAMTA1-mediated tumorigenic activity.

At the same time, the team performed a pilot in vivo experiment and found that NIH3T3-TAZ-CAMTA1 cells form tumors in mice, which suggests that they can use these cells for in vivo experiments that test if their candidates influence TAZ-CAMTA1 tumorigenic function.

The team are now planning to pursue each of the 6 genes identified above, and have focused recent effort on developing the tools needed for this. They have acquired several constructs that can be used to activate or repress each of these proteins or the pathways the team predicts they are using to regulate TAZ-CAMTA1. They have been cloning these constructs into retroviral or lentiviral vectors that will allow them to stably express each protein either constitutively, or in a doxycycline-inducible manner. As part of this, the team has also developed new versions of the TAZ-CAMTA1 retroviral vector with different antibiotic selection genes, which will give them more flexibility to design these experiments. They have now begun pursuing two of their top candidates. Their preliminary data suggests that overexpression of either of these proteins can repress TAZ-CAMTA1 activity, which could mean that activation of these proteins in EHE cells could repress TAZ-CAMTA1.

Their current focus is on understanding what is causing the difference in responses seen between these first two genes, as well as looking at other drugs that repress or activate these genes, to investigate the impact that these would have, and whether these might be possible processes that could ultimately be harnessed in order to repress TAZ-CAMTA1.  

This exciting research is progressing well and we hope to have further positive news to post in the future.