View Full Version : The ENCODE Project

09-06-2012, 11:52 PM
The stretches of DNA that we call genes are only a very small piece of what makes the body work. Much more important is the stuff in between the genes. Most of the changes that affect cancer don't lie in the genes themselves but in as many as 40 million different switches that are controlling these genes, turning them on and off in complex and subtle ways. In other words, the genome is loaded with gene controlling switches.

Each gene provides the code for a single protein. The proteins are the building blocks of cells, and the products made by the cells, from compounds called growth factors to signal-carrying chemicals. An intermediary genetic structure called RNA carries this information. That RNA generates the 40 million switches that can affect how and when many things happen within the cells.

The New York Times Gina Kolata reported that now scientists have discovered a vital clue to unraveling the many mysteries of human biolgoy. The human genome is packed with at least four million gene switches that reside in bits of DNA that once were dismissed as “junk” but that turn out to play critical roles in controlling how cells, organs and other tissues behave. The discovery has enormous implications for human health because many complex diseases appear to be caused by tiny changes in hundreds of gene switches.

The findings are the fruit of a federal project involving 440 scientists from 32 laboratories around the world, and will have immediate applications for understanding how alterations in the non-gene parts of DNA contribute to human diseases, which may in turn lead to new drugs. They can also help explain how the environment can affect disease risk. In the case of identical twins, small changes in environmental exposure can slightly alter gene switches, with the result that one twin gets a disease like cancer and the other does not.

As scientists delved into the “junk” parts of the DNA that are not actual genes containing instructions for proteins, they discovered a complex system that controls genes. At least 80 percent of this DNA is active and needed. The result of the work is an annotated road map of much of this DNA, noting what it is doing and how. It includes the system of switches that act like dimmer switches for lights, control which genes are used in a cell and when they are used, and determine whether a cell becomes a liver cell or a neuron.

The discoveries were published in six papers in the journal Nature and in 24 papers in Genome Research and Genome Biology. In addition, The Journal of Biological Chemistry published six review articles, and Science published yet another article.

Human DNA is “a lot more active than we expected, and there are a lot more things happening than we expected,” said Ewan Birney of the European Molecular Biology Laboratory-European Bioinformatics Institute, a lead researcher on the project.

In one of the Nature papers, researchers link the gene switches to a range of human diseases, multiple sclerosis, lupus, rheumatoid arthritis, Crohn’s disease, celiac disease, and even to traits like height. In large studies over the past decade, scientists found that minor changes in human DNA sequences increase the risk that a person will get those diseases.

But those changes were in the junk, now often referred to as the dark matter, they were not changes in genes, and their significance was not clear. The new analysis reveals that a great many of those changes alter gene switches and are highly significant.

“Most of the changes that affect disease don’t lie in the genes themselves; they lie in the switches,” said Michael Snyder, a Stanford University researcher for the project, called Encode, for Encyclopedia of DNA Elements.

And that, said Dr. Bradley Bernstein, an Encode researcher at Massachusetts General Hospital, “is a really big deal.” He added, “I don’t think anyone predicted that would be the case.”

The discoveries also can reveal which genetic changes are important in cancer, and why. As they began determining the DNA sequences of cancer cells, researchers realized that most of the thousands of DNA changes in cancer cells were not in genes; they were in the dark matter. The challenge is to figure out which of those changes are driving the cancer’s growth.

“These papers are very significant,” said Dr. Mark A. Rubin, a prostate cancer genomics researcher at Weill Cornell Medical College. Dr. Rubin, who was not part of the Encode project, added, “They will definitely have an impact on our medical research on cancer.”

In prostate cancer, for example, his group found mutations in important genes that are not readily attacked by drugs. But Encode, by showing which regions of the dark matter control those genes, gives another way to attack them: target those controlling switches.

Dr. Bernstein said, “This is a resource, like the human genome, that will drive science forward.” The system, though, is stunningly complex, with many redundancies. Just the idea of so many switches was almost incomprehensible, Dr. Bernstein said. There also is a sort of DNA wiring system that is almost inconceivably intricate.

There is another sort of hairball of electrical wires as well: the complex three-dimensional structure of DNA. Human DNA is such a long strand, about 10 feet of DNA stuffed into a microscopic nucleus of a cell, that it fits only because it is tightly wound and coiled around itself.

When they looked at the three-dimensional structure, Encode researchers discovered that small segments of dark-matter DNA are often quite close to genes they control. In the past, when they analyzed only the uncoiled length of DNA, those controlling regions appeared to be far from the genes they affect.

The project began in 2003, as researchers began to appreciate how little they knew about human DNA. In recent years, some began to find switches in the 99 percent of human DNA that is not genes, but they could not fully characterize or explain what a vast majority of it was doing.

The thought before the start of the project, said Thomas Gingeras, an Encode researcher from Cold Spring Harbor Laboratory, was that only 5 to 10 percent of the DNA in a human being was actually being used.

The big surprise was not only that almost all of the DNA is used but also that a large proportion of it is gene switches. Before Encode, said Dr. John Stamatoyannopoulos, a University of Washington scientist who was part of the project, “if you had said half of the genome and probably more has instructions for turning genes on and off, I don’t think people would have believed you.”

By the time the National Human Genome Research Institute, part of the National Institutes of Health, embarked on Encode, major advances in DNA sequencing and computational biology had made it conceivable to try to understand the dark matter of human DNA.

Even so, the analysis was daunting — the researchers generated 15 trillion bytes of raw data. Analyzing the data required the equivalent of more than 300 years of computer time.

“There is literally a flotilla of papers,” Dr. Gerstein said. But, he added, more work has yet to be done; there are still parts of the genome that have not been figured out.

Investigators in the field of drug response prediction know the nuances of human biology encompass the epigenetic, siRNA, pseudogene, non-coding DNA and protein kinetics that ultimately characterize the human phenotype. For all these reasons, the simple measurement of gene sequences cannot accurately predict biological behavior. One of those investigators of drug response tells us that genes do not make us what we are, they only (sometimes) permit us to become what we are, with the vagaries of transcription and translation lying between.

09-06-2012, 11:55 PM
In many ways, cancer can be viewed like an integrated circuit. On-off switches and back up switches constitute the controls. When one of these switches stops working and a cell “short circuits,” cancer is the result.

As we become more sophisticated in our understanding of cancer biology, we begin to create drugs that specifically target these short circuits.

On April 3, 2011, Dr. Robert Nagourney reported his most recent findings on novel compounds that target two parallel circuits in cancer cells. These compounds, or small molecules, disrupt the signal that drives cancer cell survival and proliferation.

While the profiles of each drug alone are of interest, the profiles of the drugs in combination are better still. The phenomenon of cross talk defines an escape mechanism whereby cancer cells blocked from one passage, find a second.

When therapists have the capacity to block more than one pathway, the cancer cell is trapped and often dies. This is what he has observed with these duel inhibitor combinations.

What is interesting is the fact that the activities cut across tumor types. Melanomas, colon cancers and lung cancers seem to have similar propensities to drive along these paths. Once again, he found that cancer biology is non-linear.

Moreover, cancers share pathways across tumor types, pathways that might not intuitively seem related. This is the beauty of the functional profiling platform — for it allows him to explore drugs and combinations that most people wouldn’t think of. It is these counterintuitive explorations that will likely lead to meaningful advances.

The Sunday experimental and molecular therapeutics poster session at the AACR 102nd annual meeting included Dr. Nagourney's presentation on signal transduction inhibitors. Using MEK/ERK and PI3K-MTOR inhibitors he explored the activities, synergies and possible clinical utilities of these novel compounds.

The findings were instructive. First, it saw a good signal for both compounds utilizing the cell function analysis of functional cytometric profiling. Second, it saw disease-specific activity for both compounds.

For the MEK/ERK inhibitor, melanoma appeared to be a favored clinical target. This is highly consistent with expectations. After all, many melanomas carry mutations in the BRAF gene, and BRAF signals downstream to MEK/ERK.

By blocking MEK/ERK, it appeared that his lab blocked a pathway fundamental to melanoma progression. Indeed, MEK/ERK inhibitors are currently under investigation for melanoma.

For PI3K inhibitors, the highest activity was observed in uterine cancers. This has interest, because uterine carcinomas are often associated with a mutation in the PTEN gene. PTEN is a phosphatase tumor suppressor that functions to block activation of the PI3K pathway.

Thus, mutations in the tumor suppressor unleash PI3K signaling, driving tumors to grow and metastasize. Blocking PI3K provided a strong signal, indicating that this approach may be very active in tumors associated with these oncogenic events.

The third point of interest in the report was, perhaps, its most important. Specifically, that the lab can explore those diseases where MEK-ERK, PI3K and mTOR signaling are less established targets. Cancers of the lung, ovary, colon or breast all manifested profiles of interest.

When he combined both pathway inhibitors in a process called horizontal inhibition, renal cell carcinoma popped up as the best target. These results, though exploratory, suggest a superior approach for drug development, allowing the lab to identify important leads much faster than the clinical trial process.

Source: Rational Therapeutics, Inc.

Poster from Rational Therapeutics Session at 2011 AACR Meeting


09-23-2012, 01:27 AM
According to the lead author of a paper published in the journal Nature, squamous cell lung cancer is a disease where there are no targeted therapies; drugs that attack genetic abnormalities. The study was part of the Cancer Genome Atlas, a project by the National Institutes of Health to examine genetic abnormalities in cancer (the genetic view of cancer).

The genetic view of cancer (genotype) may not be central to the phenotype at all. The particular sequence of DNA that an organism possesses (genotype) does not determine what bodily or behavioral from (phenotype) the organism will finally display.

Among other things, environmental influences can cause the suppression of some gene functions and the activation of others. Our knowledge of genomic complexity tells us that genes and parts of genes interact with other genes, as do their protein products, and the whole system is constantly being affected by internal and external environmental factors.

Researchers are planning a new type of testing program for squamous cell cancer that will match the major genetic abnormality in each patient with a drug designed to attack it.

The study compared tumor cells from 178 squamous cell lung cancer patients to the patients' normal healthy cells. More than 60% of the tumors had alterations in genes used to make protein and lipid kinases, enzymes that are particularly vulnerable to a new crop of cancer drugs and for which many drugs are already available or being tested in other cancers.

Kinases function like on-off switches for cell growth. When they are mutated, the switches are stuck in an "on" position. About a dozen companies have drugs that block mutated kinases. Although squamous cell cancer often have kinase mutations, cells have many kinase genes and the mutations are different in different patients. What the Cancer Genome Atlas has revealed is that every individual's cancer could be very different from another.

Also, the study found that about 3% of tumors have a gene mutation that might allow them to evade the immune system. An experimental drug that unleashes the immune system was recently tested in lung cancer patients.

What the researchers acknowledge is the challenge to put the finding to clinical use. They have to establish that the mutations in question actually are essential to the tumors' growth. There are several steps. 1. Show that if the mutated gene is added to normal cells that they turn into cancerous cells. 2. Show that if the mutated gene is added to mice that they develop squamous cell lung cancers. 3. Show that if the gene is turned off in cells grown in a laboratory, with a drug, that the cells die.

Then they need to test the drug in patients. However, if only a small percentage of patients have each of the mutations, that will pose a problem. If squamous cell patients are subdivided according to their particular gene mutations, there would be too few for a drug test within a single medical center or even several.

So the major medical centers need to form a consortium. In it, they would direct one or more studies of one mutation and one drug that might home in on the specific mutation. So even if a small percentage of squamous cell cancer patients would have that mutation, patients across the country could be in a clinical trial of a targeted drug. A patient's own doctor could administer the drug and the medical center directing the trial could analyze the data in partnership with the company that made the drug.

This was similarly done with Xalkori (crizotinib), which targets a rearranged gene in some adenocarcinoma lung cancer patients entered clinical trials for lung cancers with the rearrangement. The rearrangement was so rare, about 1,500 patients were tested to find 82 whose cancers had it. They were the ones included in the study.

Xalkori (crizotinib) was originally developed as a clinical therapy for patients who carried the CMET mutation. Serendipity led to the recognition that the responding subpopulation was actually carrying a heretofore-unrecognized ALK gene rearrangement (accidental success). Were it not for the clinical "observation" of response in patients, the investigators conducting this trial would have been unlikely to make the discoveries that today provide such good clinical responses in others. As Dr. Robert Nagourney of Rational Therapeutics puts it, "these patients and their disease entities educated the molecular biologists."

09-27-2012, 09:09 PM
Scientists say a new report in the journal Nature provides a big leap in the understanding of how different types of breast cancer differ.

(NPR) September 24, 2012 - Scientists have known for a while that breast cancer is really four different diseases, with subtypes among them, an insight that has helped improve treatment for some women.

But experts haven't understood much about how these four types differ. A new report, published online in the journal Nature, provides a big leap in that understanding.

"This paper gives us a level of detailed knowledge of breast cancer that vastly exceeds what was available before," says Matthew Meyerson, one of the paper's 348 authors. The effort is part of The Cancer Genome Atlas network, funded by the National Institutes of Health.

Meyerson and his colleagues used a half-dozen different types of analysis to comb through the genes of 825 breast cancer patients.

"We basically studied the genomes of breast cancers from each of these women in comparison to the genomes of the rest of their bodies," Meyerson told Shots.

They found 40 or so key differences in the genes among the four major types of breast cancer. The types are basal-like (also called triple-negative), luminal A, luminal B and HER2-enriched.

All of these differences are potential targets for cleverly designed drugs.

Some of the similarities between breast cancer and other types of cancer may also be important clues. For instance, the researchers found that certain mutations may underlie basal-like breast cancer and ovarian cancer, especially in women who inherit a BRCA-1 or BRCA-2 mutation.

So some think it might be reasonable to try ovarian cancer drugs in breast cancer patients with these genetic markers.

But Meyerson, who's with the Dana Farber Cancer Institute in Boston and the Broad Institute across the river in Cambridge, Mass., cautions that all of this is a long way from the outmoded dream of a silver bullet that would knock out breast cancer — or any other kind.

"I think in the end, to treat cancer, we're going to be developing a lot of specific silver bullets, but we'll need to use them in combination," he says. "So you'll really need a gold bullet and a silver bullet and a bronze bullet altogether to effectively treat cancers."

Charles Perou of the University of North Carolina, the first author on the paper, says that arsenal is being developed.

"The bad news is that it's complicated. And we have to figure out which bullet is to be used, where and when," he says. "That has to be done in rigorous clinical trials. And that is happening."

But consumer advocates caution that it's going to take a long time to find and test all these different "bullets."

"For me, I have to say, my enthusiasm around this as new and exciting is dampened by the knowledge that it's going to be many, many, many years before we see something that is clinically meaningful for patients," Karuna Jaggar of Breast Cancer Action told Shots. "In the meantime, too many women continue to be diagnosed with the disease."

Fran Visco, president of the National Breast Cancer Coalition, agrees.

"Finding targets, doing ... genomic screening, that's not the end goal," she says. "That is simply a tool, a step on the way to figuring out how to save lives.

"We have to be careful what we celebrate," Visco adds. "And we have to be careful what we consider to be a success. We are nowhere near success."

Perou says he understands their frustration.

"We're all — including myself — disappointed in the speed required to make these clinical advances," he says. "It's far easier to make a discovery than it is to translate that discovery into a clinical impact."

Perou thinks "optimistically" that the first impact from this new research could be two to five years away. It might be for the most common type — luminal breast cancer. That's because, the new paper shows, it has relatively few genetic mutations.

Unexpectedly, the researchers found that virtually all women with luminal-A breast cancers have a mutation in a cell receptor called phosphoinositide 3-kinase. There are already drugs in the works that target PI3-kinases.