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“By Julie Grisham, MS, Science Writer/Editor | Tuesday, December 18, 2012

A team from Memorial Sloan-Kettering has reported a new technique that could allow the development of more-specific, cell-based immune therapies for cancer. These types of treatments — which make use of patients’ own immune cells that have been enhanced in the laboratory — have shown some early success in the treatment of blood cancers including certain types of leukemia.

For most cancers, however, cell-based therapies have been harder to develop, in large part because it has been difficult for investigators to train immune cells to attack cancer cells without damaging normal, healthy cells in the body.

“We are getting better at working with patients’ immune cells and enhancing them so that we can get a powerful immunological response against cancer,” says Michel Sadelain, Director of Memorial Sloan-Kettering’s Center for Cell Engineering, who led the study. “The dilemma now is that we are concerned with limiting these responses and making them as targeted as possible to avoid potentially harmful side effects. This new study helps us move toward that goal.”

“As Targeted as Possible”

The treatment approach, known as adoptive cell transfer (ACT), involves engineering an immune cell called a T cell. Also called T lymphocytes, T cells are a type of white blood cell. They work by recognizing specific antigens — proteins on the surfaces of invading cells — and mounting an immune response against these invaders.

In the ACT process, T cells are removed from a patient and a gene is added to allow the T cells to recognize a certain antigen on the surface of a cancer cell. The enhanced T cells are grown in the laboratory and then infused back into the patient to seek out and attack cancer cells.

Cancer cells overproduce certain antigens, which can help T cells to recognize them, but those same antigens are often found in lower levels on healthy cells. “There are very few antigens, if any, that are found only on cancer cells,” Dr. Sadelain explains. This means that T cells engineered to recognize a certain antigen could attack normal cells that have that same antigen as well.

“Now we are bringing in a completely new concept,” he adds. “If there is no single unique antigen that is found on the surface of the cancer cell we want to target, we instead create T cells that recognize two different antigens found on the tumor cell — a signature that will be unique to that type of cancer — and only attack cells with both antigens, sparing the normal cells that express either antigen alone.”

Balanced Signaling

The new technique makes use of two kinds of receptors: chimeric antigen receptors (CARs), which allow T cells to target antigens on the surface of a tumor cell, and chimeric costimulatory receptors (CCRs), which allow T cells to recognize a second antigen.

The CAR and the CCR work together through a process known as balanced signaling, in which the presence of either antigen on its own is not enough to trigger the immune response. Only tumor cells that carry both antigens will be targeted.

In the study, which was published online December 16 in Nature Biotechnology, the team created T cells that carried receptors for two antigens found in prostate cancer cells: a CAR for an antigen called PSMA and a CCR for an antigen called PSCA. The investigators then generated mouse models of prostate cancer and infused the mice with the engineered cells. They found that the T cells attacked only tumors that carried both antigens.

“We are the first to test this concept and show that it works,” Dr. Sadelain concludes. “We plan to develop clinical trials based on this approach, although we have not yet decided whether the first study will be a trial for prostate cancer or for a different type of cancer using two other antigens. Ultimately, our goal is to create targeted immunotherapies that are both potent and safe for patients.”” – article from Memorial Sloan-Kettering

PacBio RS and 454 DNA sequencing at engencore.sc.edu

“The work suggests new gene therapy approach to some forms of deafness”

“La Jolla, CA – December 6, 2012 – For scientists who study the genetics of hearing and deafness, finding the exact genetic machinery in the inner ear that responds to sound waves and converts them into electrical impulses, the language of the brain, has been something of a holy grail.

Now this quest has come to fruition. Scientists at The Scripps Research Institute (TSRI) in La Jolla, CA, have identified a critical component of this ear-to-brain conversion—a protein called TMHS. This protein is a component of the so-called mechanotransduction channels in the ear, which convert the signals from mechanical sound waves into electrical impulses transmitted to the nervous system.

“Scientists have been trying for decades to identify the proteins that form mechanotransduction channels,” said Ulrich Mueller, PhD, a professor in the Department of Cell Biology and director of the Dorris Neuroscience Center at TSRI who led the new study, described in the December 7, 2012 issue of the journal Cell.

Not only have the scientists finally found a key protein in this process, but the work also suggests a promising new approach toward gene therapy. In the laboratory, the scientists were able to place functional TMHS into the sensory cells for sound perception of newborn deaf mice, restoring their function. “In some forms of human deafness, there may be a way to stick these genes back in and fix the cells after birth,” said Mueller.

TMHS appears to be the direct link between the spring-like mechanism in the inner ear that responds to sound and the machinery that shoots electrical signals to the brain. When the protein is missing in mice, these signals are not sent to their brains and they cannot perceive sound.

Specific genetic forms of this protein have previously been found in people with common inherited forms of deafness, and this discovery would seem to be the first explanation for how these genetic variations account for hearing loss.

Many Different Structures

The physical basis for hearing and mechanotransduction involves receptor cells deep in the ear that collect vibrations and convert them into electrical signals that run along nerve fibers to areas in the brain where they are interpreted as sound.

This basic mechanism evolved far back in time, and structures nearly identical to the modern human inner ear have been found in the fossilized remains of dinosaurs that died 120 million years ago. Essentially all mammals today share the same form of inner ear.

What happens in hearing is that mechanical vibration waves traveling from a sound source hit the outer ear, propagate down the ear canal into the middle ear and strike the eardrum. The vibrating eardrum moves a set of delicate bones that communicate the vibrations to a fluid-filled spiral in the inner ear known as the cochlea. When the bones move, they compress a membrane on one side of the cochlea and cause the fluid inside to move.

Inside the cochlea are specialized “hair” cells that have symmetric arrays of extensions known as stereocilia protruding out from their surface. The movement of the fluid inside the cochlea causes the stereocilia to move, and this movement causes proteins known as ion channels to open. The opening of these channels is a signal monitored by sensory neurons surrounding the hair cells, and when those neurons sense some threshold level of stimulation, they fire, communicating electrical signals to the auditory cortex of the brain.

Because hearing involves so many different structures, there are hundreds and hundreds of underlying genes involved—and many ways in which it can be disrupted.

Hair cells form in the inner ear canal long before birth, and people must live with a limited number of them. They never propagate throughout life, and many if not most forms of deafness are associated with defects in hair cells that ultimately lead to their loss. Many genetic forms of deafness emerge when hair cells lack the ability to transduce sound waves into electric signals.

Over the years, Mueller and other scientists have identified dozens of genes linked to hearing loss—some from genetic studies involving deaf people and others from studies in mice, which have inner ears that are remarkably similar to humans.

A Clearer Picture

What has been lacking, however, is a complete mechanistic picture. Scientists have known many of the genes implicated in deafness, but not how they account for the various forms of hearing loss. With the discovery of the relevance of TMHS, however, the picture is becoming clearer.

TMHS turns out to play a role in a molecular complex called the tip link, which several years ago was discovered to cap the stereocilia protruding out of hair cells. These tip links connect the tops of neighboring stereocilia, bundling them together, and when they are missing the hair cells become splayed apart.

But the tip links do more than just maintain the structure of these bundles. They also house some of the machinery crucial for hearing—the proteins that physically receive the force of a sound wave and transduce it into electrical impulses by regulating the activity of ion channels. Previously, Mueller’s laboratory identified the molecules that form the tip links, but the ion channels and the molecules that connect the tip link to the ion channels remained elusive. For years, scientists have eagerly sought the exact identity of the proteins responsible for this process, said Mueller.

In their new study, Mueller and his colleagues showed that TMHS is one of the lynchpins of this process, where it is a subunit of the ion channel that directly binds to the tip link. When the TMHS protein is missing, otherwise completely normal hair cells lose their ability to send electrical signals.

The scientists demonstrated this using a laboratory technique that emulates hearing with cells in the test tube. Vibrations deflected off the cells mimic sound, and the cells can be probed to see if they can transduce the vibrations in electrical signals—as they would in the body if the cells were then trying to send signals to the brain. What they showed is that without TMHS, this ability disappears.

“We can now start to understand how organisms convert mechanical signals to electrical signals, which are the language of the brain,”̈said Mueller.

In addition to Mueller, the article “TMHS is an Integral Component of the Mechanotransduction Machinery of Cochlear Hair Cells” is authored by Wei Xiong (first author), Nicolas Grillet, Heather M. Elledge, Thomas F.J. Wagner, Bo Zhao, Kenneth R. Johnson and Piotr Kazmierczak.

This work was funded with support from the National Institutes of Health (DC005965, DC007704), the Dorris Neuroscience Center, the Skaggs Institute for Chemical Biology and the Bundy Foundation.” – article from The Scripps Research Institute

PacBio RS and 454 DNA sequencing at engencore.sc.edu

“By Leah Eisenstadt, Broad Communications, December 13th, 2012

Colon cancer is one of the leading causes of cancer-related death in the United States. Risk factors for the disease are varied and include factors such as advanced age and diet, but most cases share something crucial that scientists hope can usher in new treatments. In nearly all cases, the DNA in colon tumors harbors mutations in a key intracellular process or “pathway,” leading to the buildup of proteins that drive uncontrolled growth of cells.

Because disruptions in this pathway, known as the Wnt/beta-catenin pathway, are so ubiquitous in colon cancer and other cancers, it represents a promising target for developing therapeutics. However, scientists have so far been unsuccessful in targeting the pathway. Broad Institute senior associate member William Hahn, who is also an associate professor at the Dana–Farber Cancer Institute and Harvard Medical School, describes the situation as being stuck in a room with only one exit. Scientists thought that beta-catenin had only one partner protein, a transcription factor called TCF4, through which it worked to initiate and drive cancer. With no way to target TCF4, it was as though the single door to a potential new therapy remained locked.

A new door may now be open, thanks to an ambitious effort from researchers at the Broad Institute and Dana-Farber Cancer Institute. The work, led by Hahn and made possible by the Broad’s large-scale RNA interference and cell line resources, reveals an alternative pathway through which beta-catenin drives cancer, one that may be more amenable to therapeutic targeting. The findings appear online December 13 in the journal Cell and help suggest an expanded role for proteins like beta-catenin — that they may have more than one path in the cell and more than one role in cancer.

The new study was motivated not only by the desire for new therapeutic targets, but also by the need for a deeper understanding of beta-catenin’s workings in the cell. Recent genome sequencing studies, some completed at the Broad, revealed that beta-catenin’s traditional partner protein, TCF4, was actually missing or inactivated in more than a quarter of cases of colon cancer. The absence of this key partner suggested that beta-catenin had other partner proteins — indicating potential hidden pathways through which it worked to cause cancer.

To begin the hunt for a new pathway, the team set out to find genes that were essential to the survival of cancers driven by beta-catenin, representing its potential partners in the cell. They first classified 85 cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia based on whether beta-catenin was activated (as it is in most colon cancers) or not. In parallel, they analyzed data on those cell lines from an RNA interference screen completed as part of the Cancer Program’s Project Achilles, which identified genes essential for the survival of cancer cells, the so-called “Achilles heels” of cancer.

The results pointed to genes that regulate a protein called YAP1, which controls the activity of other genes. Through extensive follow-up work in cellular and animal models, the team discovered a new complex of proteins that work together with beta-catenin to regulate the transcription, or activity, of genes that drive cancer. Further, they found that the new complex is regulated by YES1, an enzyme in the kinase family of proteins that have proven to be more amenable to therapeutic targeting than transcription factors. The team was even able to use a small molecule to inhibit YES1, blocking the activity of the complex and halting the growth of cancer cells.

“It’s a bit of a surprising finding because most of us thought that beta-catenin has one partner — TCF4 — that’s responsible for all its roles in cancer,” said Hahn. The new results help explain how cancer cells without a working TCF4 protein could still be cancerous. “By finding that there’s another set of partners that beta-catenin could have, it helps explain those findings and it may allow us to have an idea about how these complexes play different roles at different stages of tumor development.”

The discovery of beta-catenin’s new pathway was made possible by the Broad’s resources for large-scale, systematic exploration, in addition to informatics expertise. “We’ve been studying this pathway for twenty years, and we didn’t recognize that there was this other, very important component of it,” said Hahn. “It’s because we didn’t have the tools to do it until now.”

“This work demonstrates the power of Project Achilles and the advantage of using so many cell lines,” said Joseph Rosenbluh, first author on the study. “We could identify new things, simply because we have larger numbers than ever before.”

The findings reveal a new branch of the Wnt/beta-catenin pathway that’s likely important in development and cancer. In addition, targeting the pathway through the YES1 kinase may successfully prevent tumor growth in a large percentage of patients, if a safe and effective targeting therapy were developed. The team is currently looking into further studies of an existing drug that is FDA-approved for other conditions and that targets YES1. Finding a way to target the pathway could make a significant impact on the treatment of this disease, because it is altered in such a large proportion of cases. As Rosenbluh explained, “Finding a way to target beta-catenin will allow us to target all colon cancers.”

The work also demonstrates that systematic studies of the function of genes are a very good complement to those aimed at characterizing cancer genomes. “As genome sequencing in cancer and other conditions becomes cheaper and more routine, the emphasis must shift to the understanding of the function of genes,” Hahn said. “This is a good example of how we’re moving in parallel [to sequencing studies] to do that.”

The new pathway increases the complexity of beta-catenin’s role in the cell, but offers significant opportunities, too. “This complexity we’ve uncovered is probably going to get us closer to understanding how things really work,” said Hahn. “A new door has been opened that tells us there are many other ways we can understand this pathway, and that gives us new opportunities to think about intervening. YES1 is a really promising one, but it may be just the first of many things to come.”

Other Broad researchers involved in this study include Deepak Nijhawan, Eric Schafer, Travis Zack, Xiaoxing Wang, Aviad Tsherniak, Anna Schinzel, Diane Shao, Steven Schumacher, Barbara Weir, Francisca Vazquez, Glenn Cowley, David Root, Jill Mesirov, and Rameen Beroukhim.

“Gene knockout stops immune cell development“

Researchers at the Wellcome Trust Sanger Institute have identified the key gene in ensuring that our immune defences develop infection-fighting cells. No cells of the adaptive immune system – key to attacking and destroying bacteria and other pathogens – develop in the absence of the gene Bcl11a.

The result will help to understand the human immune system and how it can fail in disease, as well as possibly allowing the development of functioning human immune systems in mice for research and development of treatments.

Our immune system has two arms: the adaptive and innate pathways. The adaptive immune system leads to infection- and cancer-fighting cells called B cells, T cells and NK cells: these all share a common ancestor, or lymphoid progenitor.

The team had shown earlier that Bcl11a was important for development of some immune cells in mouse embryos. In the new research they looked at the role of this gene in adult mice. They knocked out Bcl11a and looked at development of the immune system cells.

They were surprised to see that no cells of the adaptive system developed in the mice.

“This is the cornerstone of building an effective immune system,” says Dr Pentao Liu, who led the research. “It is perhaps the first time that anyone has found a single gene that is absolutely essential for development of cells of our entire adaptive immune system.

“With this new discovery, we can understand better how our immune system is built and begin to learn how we might repair it.”

The protein produced by Bcl11a controls the activity of other genes: it is part of a network of components that regulate cell development and the imbalance caused by a lack of Bcl11a leads to the death of the progenitor cells of the adaptive immune system. By contrast, overactivity of Bcl11a is known to cause lymphomas.

“This is the most important gene in the adaptive immune system,” says PhD student Yong Yu, who did the research while at the Wellcome Trust Sanger Institute. “Without it, there are no cells of this system, no antibodies to fight infection.

“We have begun to tease apart the networks that have to work in balance to give us a healthy immune system.”

One member of the Bcl11a network is a gene called p53, known to be important in controlling cell division and, when mutated, important in driving cancer development. If p53 also is inactivated in mice that lack Bcl11a, some cells of the immune system develop. Uncovering these interactions will drive a better understanding of disease of the immune system.

Mice that lack functional Bcl11a and hence lack a functional immune system could be important in studying human cells and human immune biology. By adding human disease cells to the immune-deficient mice, researchers could define the function of human genes in a humanised environment in order, for example, to examine the role of genes in transplant biology.

Bcl11a also has a role in development of oxygen-carrying red blood cells: in 2011, researchers showed that silencing Bcl11a in mice could reverse sickle-cell disease.

“Our discovery shows that Bcl11a has a central and essential role in the immune system, alongside its other functions,” continues Dr Liu. “It is work that our mouse models have driven. Understanding the complex interactions of networks of genes in this way will be essential for better understanding human disease and in finding better ways to diagnose and treat patients.

“We already see that Bcl11a is involved in lymphoma and anaemia. We can now look to see if it has a role in other human disease.” – article from The Wellcome Trust Sanger Institute

PacBio RS and 454 DNA sequencing at engencore.sc.edu

Salk findings may help explain connections between dietary schedules and chronic disease

“La Jolla, CA—When it’s dark, and we start to fall asleep, most of us think we’re tired because our bodies need rest. Yet circadian rhythms affect our bodies not just on a global scale, but at the level of individual organs, and even genes.

Now, scientists at the Salk Institute have determined the specific genetic switches that sync liver activity to the circadian cycle. Their finding gives further insight into the mechanisms behind health-threatening conditions such as high blood sugar and high cholesterol.

“We know that genes in the liver turn on and off at different times of day and they’re involved in metabolizing substances such as fat and cholesterol,” says Satchidananda Panda, co-corresponding author on the paper and associate professor in Salk’s Regulatory Biology Laboratory. “To understand what turns those genes on or off, we had to find the switches.”

To their surprise, they discovered that among those switches was chromatin, the protein complex that tightly packages DNA in the cell nucleus. While chromatin is well known for the role it plays in controlling genes, it was not previously suspected of being affected by circadian cycles.

Panda and his colleagues, including Joseph R. Ecker, holder of the Salk International Council Chair in Genetics, report their results December 5, 2012 in Cell Metabolism.

Over the last ten years, scientists have begun to discover more about the relationship between circadian cycles and metabolism. Circadian cycles affect nearly every living organism, including plants, bacteria, insects and human beings.

“It’s been known since the early eighteenth century that plants kept in darkness still open their leaves in 24 hour cycles. Similarly, human volunteers also maintain circadian rhythms in dark rooms. Now we’re determining the regulatory processes that control those responses,” says Ecker, who was recently elected a fellow of the American Association for the Advancement of Science for his work on the genetics of plant and human cells.

Panda offers an example of human circadian influenced behavior that is painfully familiar to all parents of newborns: Why do infants wake up in the middle of the night? It isn’t because they aren’t yet “trained” to a regular schedule, but because their internal circadian clocks haven’t even developed.

“Once the clock is developed, the infant can naturally sleep through the night,” Panda says. “On the other end of the scale, older people with dementia have sleep problems because their biological clock has degenerated.”

In the case of humans and other vertebrates, a brain structure called the suprachiasmatic nucleus controls circadian responses. But there are also clocks throughout the body, including our visceral organs, that tell specific genes when to make the workhorse proteins that enable basic functions in our bodies, such as producing glucose for energy.

In the liver, genes that control the metabolism of fat and cholesterol turn on and off in sync with these clocks. But genes do not switch on and off by themselves. Their activity is regulated by the “epigenome,” a set of molecules that signal to the genes how many proteins they should make, and, most importantly from the circadian point of view, when they should be made.

“We know that when we eat determines when a particular gene turns on or off, for example, if we eat only at nighttime, a gene that should be turned on during the day will turn on at night,” Panda says.

For this reason, the epigenome is of particular interest for health, since we can control when we eat. An earlier study from Panda’s lab, published last May in Cell Metabolism, suggested that we should observe a 16-hour fast between our evening and morning meals.

“In response to natural cycles, our body has evolved to make glucose at nighttime,” Panda says. “But if on top of that you eat, you’re creating excess glucose and that damages organs, which leads to diabetes. It’s like over-charging a car battery. Bad things will happen.”

In short, while we can’t control what genes we’re born with, we do have some influence over what they do. Nevertheless, the interplay between genome and epigenome is extremely complex. Panda, Ecker and their colleagues, including the paper’s co-first authors Salk postdoctoral researchers, Christopher Vollmers and Robert J. Schmitz, did their studies in mice. In the mouse liver, they discovered more than 3,000 epigenomic elements, which regulate the circadian cycles of 14,492 genes. Comparing the mouse genome to the human genome, they find many of the same genes.

“Now that we know where the switches are, it brings us one step closer to understanding the mechanism of gene regulation,” says Panda, “For example, it helps us restrict our search for other factors to particular regions of the genome. In other words, at least we now know to search in Alaska, rather than Australia. But Alaska’s still a big place.”" – article from the Salk Institute for Biological Studies

PacBio RS and 454 DNA sequencing at engencore.sc.edu