One of the biggest breakthroughs so far in the war on cancer was the realization that it is essentially a genetic disease. However, as we learn more about cancer, it becomes clearer that what’s written in our DNA is only part of the story; there are other factors at work that go beyond genetics.
It is on this latter point that a new area of research, known as epigenetics, is building upon our knowledge of the genetics of cancer. Epigenomics, defined broadly, is the study on how the genome responds to the environment and how this response can have a long-term impact on the activity of our genes. To understand what epigenomics is one must first start with the definition of the term “genome”: the entire DNA content of a cell. In contrast, the term “epigenome” refers to chemical modifications of the DNA itself and of proteins that control the structure and activity of the genome. The genome remains mostly the same throughout an individual’s life, whereas the epigenome changes dramatically – but in very precisely controlled ways – during normal development and aging. Deviations from these normal changes in the epigenome can have profound effects on which genes are turned on and hence can cause cells to behave abnormally. More specifically, they can cause the onset of cancer.
Canada’s Michael Smith Genome Sciences Centre (GSC), a department of the BC Cancer Agency (BCCA) in Vancouver, Canada, is very active in exploring how to use epigenomics as a tool against cancer. In early 2012, the GSC launched the CEEHRC (“Canadian Epigenetics, Environment and Health Research Consortium”) epigenomics platform and data coordination centre, funded by the Canadian Institutes of Health Research and Genome BC.
Since its early days in 1999 the GSC, under the leadership of Dr. Marco Marra, has steadily grown and now occupies 50,000 sq. ft. and currently employs approximately 310 staff members. In addition to its core services the GSC has created a team of scientific, technical and project management staff to develop and operate a world class epigenome mapping pipeline founded on the Illumina massively parallel sequencing platform over the last six years. The centre operates several genome-scale cores, including a biospecimen core; a sequencing core; a library core; an engineering core; a technology development core; a quality assurance core; an informatics core; and a systems core. Each core has a group lead, and is staffed by skilled and experienced technical and scientific staff who work together to operate critical infrastructure, as well as plan and execute experiments.
As the head of epigenomics at the GSC, Dr. Martin Hirst is currently involved in several cancer-specific epigenetic projects.
“I’ve always been interested in transcriptional regulation, the study of how genes are turned on and off,” Dr. Hirst explains. His entry into the field of epigenomics in 2006 coincided with the introduction of a revolutionary DNA sequencing technology, massively parallel sequencing.
According to Dr. Hirst, before the arrival of these devices, there really was no way of doing large scale or whole genome epigenetic studies.
“The DNA sequencing platform was then called the Solexa 1G Sequencer (since acquired by Illumina Inc.), and it held great promise because it enabled the sequencing of millions of short DNA fragments simultaneously. At the time of its introduction, the platform was capable of generating 10s of millions of short DNA sequences – ideally suited for the sequencing of epigenomic libraries – in a sense it gave birth to the field of epigenomics.”
Currently, there are 11 Illumina HiSeq 2000 platforms, two Illumina HiSeq 2500s, three Illumina miSeqs and one Ion Torrent Personal Genome Machine performing sequencing at the GSC. Sequencing capabilities include both single-end (50 and 75 base read lengths) and paired-end sequencing (50, 75 and 100 base read lengths). The GSC has to date constructed over 25,000 libraries and generated over 267 terabases (2.67 x 1014) of data on the Illumina sequencing platform, contributing to numerous national and international projects and high impact publications.
Using this technology, Dr. Hirst and his collaborators are piecing together how transient and stable chemical modifications to our DNA and associated proteins (known collectively as our epigenome) cause our genome to stay healthy or develop diseases. Initially, the researchers are investigating changes to tissues and cells that lead to cancers such as leukemia, as well as cancers of the colon and ovaries, all of which are the most common human malignancies.
“Going back now almost three decades, researchers have been examining epigenetic phenomena in the development and progression of cancer, and we now know that cancer can be defined by both genetic and epigenetic disruptions,” says Dr. Hirst
He explains that what this means is that these genetic and epigenetic alterations are intimately linked to one another. One example he gives is a chemical modification of DNA called methylation.
“In nearly all cancers that have been studied this mark is dramatically reduced in the malignant cell population. In addition, imbedded within this genome-wide loss of DNA methylation there are specific regions, called CpG islands, that become hypermethylated, and can affect the control of nearby genes. For example, there are cases of tumour suppressors that are genetically mutated, so they become inactive. In the context of breast cancer that would be BRCA1. BRCA1 can also be inactivated through DNA methylation, so this epigenetic modification that occurs actually shuts the gene down and results in the loss of the tumour suppressor protein. Just as a genetic mutation of the gene would inactivate the gene because it’s nonfunctional, it can be shut off through epigenetic mechanisms as well. And there is also a sort of collusion where it seems that in certain cases, one of the gene copies (called alleles) can be genetically inactivated and the other allele, becomes methylated and thereby turned off. So it really does seem as though the genetic and epigenetic phenomena are really intertwined. And finally in many cancers, in particular in hematopoietic malignancies or blood cancers, what’s come out in the last three or four years of deep genome sequencing, is that many of them contain genetic mutations in the genes which encode for proteins that regulate the epigenome.”
The goal is to understand how these enzymes behave, how they affect the development of cancer, with an ultimate goal of applying these findings to develop new diagnostics and therapeutics. Specifically, if an enzyme adds or removes too many modifications, and thereby causes cancer, you can try to inhibit that enzyme.
One remarkable finding that has come about from this field of research, says Dr. Hirst, is that epigenome changes are reversible. Several inhibitors of chromatin-modifying enzymes, including histone deacetylase and DNA methyltransferase inhibitors, have demonstrated clinical anti-cancer activity. Dr. Hirst explains that this new understanding of how cancers work has the potential to yield new treatments.
“There are clinical trials using epigenetic based therapeutics, for example, DNA methylation inhibitors are being used in the clinic to treat certain types of leukemia, and the thought is that by inhibiting DNA methylation, you can release that repressive signal that’s present on tumour suppressor genes, allowing those genes to then be transcribed and then those genes can tell the cell things have gone wrong.”
The GSC-BCCA team has had its own success story around one such enzyme: EZH2. The GSC-BCCA team discovered a mutation in this gene, which had not been previously associated with cancer, in certain kinds of lymphoma. These findings were published in 2010 in the international journal Nature Genetics.
“It was a key finding and one that emphasizes the highly collaborative work that defines the GSC. The initial finding would not have been possible without a close collaborate on with clinical colleagues at the BC Cancer Agency, Drs. Gascoyne and Connors, who provided primary cancer tissue for molecular profiling. Building on this discovery are groups led by Dr. Humphries at the Terry Fox Laboratory, developing mouse models to study the functional consequences of the mutation and Dr. Aparicio working on inhibiting the mutant enzyme.”
Not surprisingly, working with clinicians is a critical component of what scientists at the GSC do.
“Clinicians are our partners and collaborators, who provide invaluable expertise and access to primary human tumour samples. Without these critical connections we would not be able to perform these epigenetic studies. These collaborations are an essential component of translational research, helping to drive emerging technologies into the clinic.”
The discovery of the role of EZH2 has directly led to companies pursuing an inhibitor to EZH2, with the hope of having some efficacy in the treatment of certain types of lymphoma.
In addition to its work understanding enzymes, the GSC is also developing new applications for massively parallel sequencers.
“The technique that I started with in terms of my entry into epigenomics for example, is a technique called ChIP (Chromatin Immunoprecipitation) sequencing. That was not a technique that the company had even considered for that application – they were focused on genome sequencing.”
Another application that Dr. Hirst and the GSC team helped develop was RNA sequencing. His team has created new protocols for what is called mRNA-seq. It turned out to be an important tool for studying the role of EZH2 mutations.
In addition to his work with the GSC, Dr. Hirst also operates out of his lab, the Laboratory of Epigenomics and Chromatin Biology at the Centre for High-Throughput Biology at the University of British Columbia. The work conducted at UBC focuses on developing technologies to make the sequencing of the epigenome more efficient. One of the key limitations with current sequencing technologies is that when sequencing an epigenome, significant amounts of material like tissues or cells are needed. Dr. Hirst’s goal is to reduce the amount of input material so that researchers will be able to analyze the genome or the epigenome from small numbers of cells with the ultimate go of profiling single cells.
“This entails developing new assays and working with other basic scientists at UBC, and then transferring these methodologies to the GSC, which is taking those developed protocols and applying them to a production environment in the context of primary human material.”
Dr. Hirst is also part of a number of initiatives with goals to deliver a complete reference of epigenome information on a full spectrum of normal and malignant mammalian cell types; develop novel molecular and computations technologies for broader application of epigenomic research in the future; and characterize the impact of epigenetic modifier mutations emerging from international cancer genome sequencing efforts. This includes generating highresolution reference epigenome maps that can be exploited to provide new insights into many diseases and finding new means to control them.
In Canada, the GSC is one of the two Canadian epigenome mapping and data centres under the umbrella of the Canadian Epigenetics, Environment and Health Research Consortium. In addition, the GSC also contributes, as the only foreign member institution, to the NIH Reference Epigenome Consortium.
“We work with Dr. Costello from the University of California, San Francisco (UCSF) who leads one of the four roadmap centres. We provide epigenetic mapping expertise to that project. We have been working on this project for four years and have generated reference epigenomes for various brain, breast, blood, skin, embryonic and now working on placenta cell types.”
“Large scale human reference epigenome mapping which was initiated by the NIH reference epigenome mapping consortium, has now been partnered with initiatives in Canada, (CEEHRC), a European project called Blueprint (a large-scale European research project that includes 41 leading European universities, research institutes and industry entrepreneurs) as well as projects in Germany, Italy, South Korea and Japan who are working together as part of the International Human Epigenome Consortium to generate 1,000 human reference epigenomes over the next five years.”
With his expertise in epigenomic mapping, Dr. Hirst is chairing the International Human Epigenome Consortium Assay Standards working group.
“You can think of this in the same way as one would think of the reference human genome project, which has revolutionized human medicine. What we are attempting to do is define references for the many cell types that are present in the body, to provide baseline data for the study of variations that occur normally in the human population and alterations that occurs as a consequence of disease.”
Unlike the reference human genome, where the genome is for the most part the same throughout the body, the epigenome is actually cell type specific. As such, the context of the International Human Epigenomic Consortium is to provide replicated references for approximately 200 human cell types.
“That is why we need 1,000 reference epigenomes and not just one as was the case for the genome. We will need to capture the breadth of different cell types present in the human body. Where will it go from there, I would imagine the next phase will involve understanding variation present in the human population. One exciting and largely unexplored area is how environmental signals are patterned in the epigenome. Model organism and epidemiological studies have shown that environmental cues such as stress, maternal care, nutrients, toxins etc. seem to be patterned through the epigenome. And these patterns can be inherited. It is as if the epigenome is providing shorter-term plasticity to respond to environmental changes much as the genome does on much longer time scales. That’s one of the many exciting aspects of epigenetic research in general.”
Dr. Hirst is excited by the promise of what might arise from these collaborations.
“I think it’s a very exciting time to be part of this field because we are at the dawn of being able to read the epigenome and this will lead to greater understanding of how the epigenome contributes to normal and diseased states.”
And as was the case with the EZH2 enzyme, he foresees that future discoveries in epigenomics will aid in the war on cancer.