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Breast Cancer Clue
Inherited mutations in the BRCA1 or BRCA2 tumor suppressor genes are by far the most frequent contributors to hereditary cancer risk in the human population, often causing breast or ovarian cancer in young women of child-bearing age. Attempts to test the role that the BRCA genes play in regulating a repair process associated with genome duplication have proven frustratingly difficult in living mammalian cells.
Now Harvard Medical School investigators at Beth Israel Deaconess Medical Center report a new mechanism by which BRCA gene loss may accelerate cancer-promoting chromosome rearrangements. The new findings explain how the loss of BRCA1 or BRCA2 function impairs homologous recombination, a normally accurate repair process used to fix DNA breaks, and actually stimulates faulty, error-prone homologous recombination repair.
Described online in the April 28 issue of Nature, the discovery could ultimately provide clinicians with valuable new information to help them ascertain risk and guide patient treatment when faced with BRCA mutations of uncertain significance. The finding also offers a potentially valuable new tool for the development of cancer therapeutics.
“Mutations in the BRCA genes cause breast and ovarian cancers that affect thousands of women throughout the U.S. and around the world, often striking them in the prime of life,” said senior author Ralph Scully, HMS associate professor of Medicine at Beth Israel Deaconess in the hospital’s Breast Cancer Oncology program. “For almost two decades, scientists have been striving to better understand the tumor suppressor functions of BRCA1 and BRCA2.”
Potentially harmful breaks in DNA strands commonly occur during DNA replication, a prerequisite for cell division. These breaks occur when the replication fork that duplicates the genome stalls at sites of DNA damage. If not properly repaired, the breaks can promote genomic instability, leading to cancer and other diseases.
“Some years ago, we and others suggested that BRCA1 and BRCA2 regulate homologous recombination at sites of stalled replication,” explains Scully. “We believe that this function is critical to how these genes suppress breast and ovarian cancer. Until now, we haven’t had the tools necessary to study in molecular detail the homologous recombination processes at sites of replication fork stalling in the chromosomes of a living mammalian cell.”
To solve this problem, first author Nicholas Willis, HMS research fellow in medicine in the Scully laboratory, created a new tool by harnessing a protein-DNA complex that evolved in bacteria.
“We found that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal homologous recombination in mouse cells,” explained Willis. “In its essence, E. coli bacteria—a standard model organism in science—has evolved a very simple system to arrest replication forks in a site-specific manner.”
This system is composed of short DNA elements called Ter sites, which are 21 to 23 base pairs long and tightly bound by the protein Tus. “Tus binds these Ter elements with extremely high affinity and, upon replication fork approach, acts as a barrier to fork progression along the DNA. Tus/Ter effectively sets up a ‘roadblock’ and stalls the replication fork.”
The acid test for the new tool, said the authors, came when this same short Ter sequence was placed into a reporter, a slightly larger DNA sequence that can undergo certain rearrangements within a chromosome when triggered to do so. “When it engaged in homologous recombination, a change in the sequence caused the cells to express green fluorescent protein,” said Willis. “When the cells glowed green, we knew we had a positive event.”
The team adapted the reporter sequence to distinguish between error-free/high-fidelity homologous recombination and an error-prone/aberrant form of homologous recombination. “Remarkably, when we studied cells lacking BRCA1 or BRCA2, we found that the frequency of aberrant homologous recombination events triggered at Tus/Ter-stalled replication forks had actually increased compared to normal cells. We knew at this point that we had discovered a new and important process by which BRCA gene loss promotes cancer.”
The discovery provides a promising bridge between basic science and the clinic, said Scully. “Sometimes a genetic sequencing test reveals a mutation in BRCA1 or BRCA2 that has not been definitively associated with cancer,” he said. Often described as “variants of uncertain significance,” these mutations are not found in high enough frequency in healthy women or in women with breast or ovarian cancers to allow the specific BRCA1 or BRCA2 mutations to be reliably classified as high risk or low risk. This is an important issue because a woman with a known high-risk BRCA gene mutation may elect to undergo potentially lifesaving prophylactic mastectomy or oophorectomy, Scully said.
“There is a growing appreciation that careful measurement of the homologous recombination functions of BRCA1 and BRCA2 variants-of-uncertain-significance mutants might help to classify them into high-risk or low-risk groups,” he said. “It would be gratifying if our system could contribute new information to help ongoing efforts to classify these mutants.”
Furthermore, understanding the mechanisms that regulate homologous recombination at stalled replication forks might hold additional promise for the development of novel cancer therapeutics. “If we could use this tool to help develop new cancer therapies, it would be a grand slam,” said Scully. “This new system might also be useful in genome editing, which is considered a groundbreaking technology used for the development of new gene therapies.”
This study was funded, in part, by National Institutes of Health grants R01CA095175, R01GM073894, R21CA144017, and R37GM26938; by National Institutes of Health /National Cancer Institute postdoctoral fellowship 5T32CA081156; and by ACS postdoctoral fellowship PF-12-248-01-DMC.
Adapted from a Beth Israel Deaconess news release.
Aspirin Tied to Lower Colon Cancer Risk—for Some
The humble aspirin may have another beneficial effect beyond easing the pain of headache and reducing the risk of heart attack: lowering colon cancer risk among people with high levels of a specific enzyme.
The finding comes from a multi-institutional team that analyzed data and samples from two long-term studies involving nearly 128,000 participants. The researchers discovered that people who took aspirin and had high levels of a specific enzyme in their colons had half the risk of developing colorectal cancer compared with people who also took aspirin but whose colons showed low levels of the enzyme, called 15-hydroxyprostaglandin dehydrogenase, or 15-PGDH for short. About half of the population possesses high levels of 15-PGDH.
The results appear in the April 23 edition of Science Translational Medicine. While previous trials and prospective studies have shown that aspirin use reduces colorectal cancer risk, this retrospective study may explain why aspirin benefits some people, but not others.
The research team included scientists from Brigham and Women’s Hospital, Case Western Reserve School of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Massachusetts General Hospital and University Hospitals Case Medical Center.
“If you looked at the folks from the study who had high 15-PGDH levels and took aspirin, they cut their risk of colon cancer by half,” said senior author Sanford Markowitz, Ingalls Professor of Cancer Genetics at Case Western Reserve School of Medicine. “If you looked at the folks from the study who were low for 15-PGDH, they did not benefit at all from taking aspirin. These findings represent a clean Yes-No about who would benefit from aspirin.”
According to the American Cancer Society, colorectal cancer is the second leading cause of cancer-related deaths in the United States, with predictions that 137,000 Americans will develop the disease and 50,000 will die from it in 2014.
In the current study, the scientists built on earlier research indicating that regular use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, reduces the risk of developing colon cancer for some but not all individuals. Markowitz, also a medical oncologist at University Hospitals Case Medical Center, joined co-senior author Andrew Chan, HMS associate professor of medicine and a gastroenterologist at Mass General, to explore whether the presence of 15-PGDH led to different outcomes in developing colon cancer.
They hope to develop a test to guide physicians and patients in determining whether aspirin would help them.
The team examined tissues of 270 colon cancer patients who were among 127,865 participants followed for more than 30 years in the Harvard-based Nurses’ Health Study and Health Professionals Follow-up Study. Previous reports from the Mass General and Dana-Farber team showed that participants in these studies who regularly took aspirin had a lower risk of colorectal cancer. In earlier research, Case Western Reserve investigators and Monica Bertagnolli, HMS professor of surgery at Brigham and Women’s, had found that 15-PGDH appeared to enhance the ability of celecoxib, an anti-inflammatory medication commonly known as Celebrex, to prevent colon tumors in mice and in 16 humans tested. But when 15-PGDH was low or absent, celecoxib did not prevent colon tumors in mice or humans.
In the latest study, the investigators combined forces in a larger study to examine whether 15-PGDH levels might also be associated with the colon cancer-preventing benefits of aspirin, which lacks the adverse cardiovascular side effects of celecoxib.
The Mass General and Dana-Farber team dissected normal colon tissue from the pathology specimens of participants who developed colon cancer over the studies’ follow-up periods. The team at Case Western Reserve then analyzed these colon tissues to identify which among them had high or low levels of colon 15-PGDH. The investigators at Mass General and Dana-Farber examined how participants’ aspirin use and levels of 15-PGDH might be related to the risk of colorectal cancer.
The study’s results could lead to a test that would allow more personalized decisions about treatment to prevent colorectal cancer. People whose 15-PGDH levels indicate aspirin would have little benefit might choose to avoid the potential gastrointestinal side effects—such as stomach ulcers—that can accompany aspirin use. The researchers plan to develop a cost-effective and accessible test for measuring 15-PGDH in the colon. Chan and Markowitz believe such a test could become part of current medical practice.
“During a colonoscopy, a gastroenterologist could easily and safely take an additional biopsy from the colon in individuals for whom preventive aspirin treatment might be appropriate,” Chan said.
“There would be no reason why a good hospital pathology laboratory could not establish the test for 15-PGDH,” Markowitz said.
To confirm their findings, the study authors hope to launch a randomized, prospective clinical trial in which high-risk patients would be identified, treated with aspirin or a placebo, and monitored for development of colorectal tumors.
The mechanisms of action in 15-PGDH and in aspirin make them key players in colon cancer, the scientists said. Prostaglandins promote development of colon cancer. Aspirin helps prevent colon cancer development by blocking prostaglandins from being generated, while 15-PGDH helps prevent colon cancer development by catalyzing a reaction that “chews up” prostaglandins. Markowitz refers to the gene that produces 15-PGDH as the body’s genetic form of aspirin. The study suggests that both aspirin and 15-PGDH must work together to effectively prevent colon cancer, bringing the most benefit to individuals who have high levels of 15-PGDH.
“This study highlights the benefits of the relatively new practice of molecular pathological epidemiology, ” said co-senior author Shuji Ogino, HMS associate professor of pathology at Brigham and Women’s and Dana-Farber. “The molecular pathology part relates to analysis of 15-PGDH gene expression level in the normal colon to classify cancer based on molecular pathogenesis, while the epidemiology part relates to collection and analysis of aspirin use data in a population. This is an integration of these analyses."
Other researchers involved in the study include first author Stephen Fink, an instructor at Case Western Reserve; co-authors Mai Yamauchi and Reiko Nishihara, HMS research fellows at Dana-Farber; and senior author Charles S. Fuchs, HMS professor of medicine at Dana-Farber.
The study was supported by the Entertainment Industry Foundation’s National Colorectal Cancer Research Alliance; the National Cancer Institute’s GI-SPORE program (Specialized Programs of Research Excellence in Gastrointestinal Cancers) and Early Detection Research Network; National Institutes of Health grants P01 CA87969, P01 CA55075, 1UM1 CA167552, R01 CA136950, P50 CA127003, R01 CA151993, P50 CA150964, U01 CA152756, R01 CA137178 and K24 DK 098311; the Damon Runyon Cancer Research Foundation; and gifts from the Cleveland-based Marguerite Wilson Foundation, the Leonard and Joan Horvitz Foundation, the Richard Horvitz and Erica Hartman-Horvitz Foundation, and the Boston-based Bennett Family Fund for Targeted Therapies Research.
Adapted from a joint Case Western Reserve and Mass General news release.
Genes and Galaxies
If you haven’t thought about reworking the human genome so people can colonize other planets, don’t worry. Plenty of people are on it.
Scientists of many stripes have been figuring out what barriers would keep us from calling distant, inhospitable galactic real estate “home” if—or when, depending on your point of view— we damage the Earth enough to face extinction. And then there’s the whole question of whether we should try to win a stay of execution for our species. After all, what makes us so special?
Those questions were just the beginning of a free-form symposium hosted March 13 by the HMS Department of Genetics on “Genetics, Biomedicine, and the Human Experience in Space,” the standing-room-only crowd in attendance fueled by pizza and unbridled curiosity.
Speakers quickly made clear why space travel and exploration over vast, uncharted distances depends on numerous, unknown factors hidden in our genes. Living with microgravity while being bombarded with cosmic rays can affect different people different ways. Scientists want to know why—and which genes might make it better or worse.
Also, space is just cool.
The Role of Genetics
The session unleashed uninhibited discussion, with a fairly even split between prepared presentations and informed thinking-out-loud improv from the audience.
“We are a medical school. Whether or not you agree with sending people into space, we are responsible for their health on and off the planet,” said Wu.
Well-known muscle and skeletal weakness and sleep disruption are not the only problems humans encounter in space. Physical concerns ride along with behavioral and neuropsychiatric issues aboard current spacecraft, not to mention whatever vehicles might ferry people farther away. It’s lonely out there.
Thinking about travel to Mars, one of our nearest neighbors, is daunting for robots, much less people. Just ask symposium guest Adam Steltzner, mechanical systems lead at the Jet Propulsion Lab, about the prodigious work that brought back what we know about the planet. Or Dorit Donoviel, deputy chief scientist and industry forum lead of the National Space Biomedical Research Institute, and assistant professor in the Department of Pharmacology and Center for Space Medicine at Baylor College of Medicine, who studies astronauts and the challenges they face, including problems with vision and headaches.
Focusing on Space
HMS geneticists spoke about the intersections between their scientific focus and space. Susan Dymecki said she began thinking about why cosmonauts in the former Soviet Union’s space program were forbidden from playing chess on board space flights. The answer involves aggression and impulsivity.
For HMS professor of genetics David Sinclair, this intersection involves the potential advantages of extant human variation and rallying our genetics to counter aging during long-distance travel spanning hundreds of thousands of years.
Bruce Yankner, HMS professor of genetics, talked about protecting the brain and memory in space. Wu presented her vision for using ultraconserved elements, which some consider to be among the most mysterious sequences of the human genome, to orchestrate chromosome behavior
to and thus protect genomes against cosmic radiation in space.
Mary Bouxsein, a biomechanical engineer and HMS assistant professor of orthopedic surgery at Beth Israel Deaconess Medical Center, a last minute addition to the program, showed the devastating effects of space flight on bone, and how that might be prevented in space—and on Earth -- with a newly developed therapy.
Genetics professor Gary Ruvkun, whose talk was entitled “What’s true for E. coli is true for the elephant” and our speculative kin on Gliese 667 Cf, (a potential Class M planet in the Gliese 667 star system), peppered the meeting with a positive view of the extinction of the human species and then proposed that, rather than travel to another planet, we “print” ourselves there. Conversely, we could print extraterrestial life on Earth.
George Church, the Robert Winthrop Professor of Genetics, suggested using genomics to identify and engage human protective variants, speculating that, by ridding ourselves of our microbiome and taking advantage of variants that suppress pain, we might create a habitat in which surgeries can occur without anesthesia or need for sterilization.
Space is vast, cold and hard for us humans, and outside of Earth, its planets, and moons—too hot, too cold, too toxic for life that evolved here— are not much more welcoming. Should we want to go there, and decide who is best suited to do so, a great deal more work needs to be done not only in jet propulsion but in human genetics.
Location, Location, Location
In biology, as in real estate, location matters. Working copies of active genes -- called messenger RNAs or mRNAs -- are positioned strategically throughout living tissues, and their location often helps regulate how cells and tissues grow and develop. But to analyze many mRNAs simultaneously, scientists have had to grind cells to a pulp, which left them no good way to pinpoint where those mRNAs sat within the cell.
Now a team at the Wyss Institute of Biologically Inspired Engineering at Harvard University and Harvard Medical School, in collaboration with the Allen Institute for Brain Science, has developed a new method that allows scientists to pinpoint thousands of mRNAs and other types of RNAs at once in intact cells -- all while determining the sequence of letters, or bases, that identify them and reveal what they do.
The method, called fluorescent in situ RNA sequencing (FISSEQ), could lead to earlier cancer diagnosis by revealing molecular changes that drive cancer in seemingly healthy tissue. It could track cancer mutations and how they respond to modern targeted therapies, and uncover targets for safer and more effective ones.
The method could also help biologists understand how tissues change subtly during embryonic development -- and even help map the maze of neurons that wire the human brain. The researchers reported the method in today’s online edition of Science.
“By looking comprehensively at gene expression within cells, we can now spot numerous important differences in complex tissues like the brain that are invisible today,” said George Church, a professor of genetics at Harvard Medical School and a core faculty member at the Wyss Institute. “This will help us understand like never before how tissues develop and function in health and disease.”
Locking RNAs in Place
Healthy human cells typically turn on nearly half of their 20,000 genes at any given time, and they choose those genes carefully to produce the desired cellular responses. Moreover, cells can dial gene expression up or down, adjusting to produce anywhere from a few working copies of a gene to several thousand.
But simultaneously pinpointing the cellular location of all those mRNAs is a tall order.
Church and Je Hyuk Lee, a research fellow at Harvard Medical School and the Wyss Institute, were up for the challenge. Moreover, they wanted to simultaneously determine the sequence of those RNAs, which identifies them and often reveals their function.
Lee and his colleagues first treated the tissue chemically to fix the cell’s thousands of RNAs in place. Then they used enzymes to copy those RNAs into DNA replicas, and copy those replicas many times to create a tiny ball of replica DNA fixed to the same spot.
They managed to fix and replicate thousands of the cell’s RNAs at once -- but then became a victim of their own success. The RNAs were so tightly packed inside the cell that even a tricked-out microscope and camera could not distinguish the flashing lights of one individual ball of replica DNA from those of its neighbors.
A new way to image RNA
To solve that problem, the researchers pioneered an unconventional method to visualize tiny objects inside cells. It works like an urban postal system. If a postmaster tried to identify each home in her city by color, she would quickly run out of colors as new homes were built, resulting in undelivered mail. Instead, postmasters keep track of each home by assigning it a unique address.
The researchers realized they could assign each RNA in the cell a unique address: the sequence of “letters,” or bases, in the RNA molecule itself. They figured they could read the address using methods akin to next-generation DNA sequencing, a set of high-speed genome sequencing methods Church helped develop in the early 2000s.
In next-gen sequencing, scientists grind up tissue, extract its DNA, break the DNA into pieces, then dilute those pieces enough so that each piece of DNA sticks to a separate spot on a glass slide. They use enzymes and four different fluorescent dyes -- one each for each of the four “letters,” or bases -- to make the DNA flash a sequence of colors that reveals its sequence.
By analogy, the scientists sought to fix RNA in place in the cell, make a tiny ball with many matching DNA replicas of each RNA, then adapt next-gen DNA sequencing so it worked in fixed cells. The four flashing colors would reveal the base sequence of each replica DNA, which would tell them the base sequence of the matching RNA from which it was derived. And those sequences would in theory provide an unlimited number of unique addresses – one for each of the original RNAs.
The scientists struggled at first to visualize the flashing lights of individual balls of replica DNA from a distance where the whole landscape of the tissue remained in view.
They succeeded by selectively turning on just a fraction of those flashing dots at any given time, so they could distinguish single balls of replica DNA flashing across the cellular landscape.
The strategy would only work, however, if they could actually read enough of the base sequence to provide a unique address. At first they could not determine more than six bases in the replica DNA, which did not provide enough unique addresses to identify individual genes in the human genome. That’s when Evan Daugharthy, a graduate student at Harvard Medical School, stepped in.
FISSEQ at Work
Daugharthy first devised an algorithm to locate the sequence of the replica DNA with the known sequence of genes in the human genome. Flashing lights that did not correspond to a real gene were erased from the image.
Then Daugharthy hacked a commercial DNA sequencing kit, which enabled the team to sequence 30 bases, more than enough to provide each replica DNA with a unique address. In this way the team could create a composite image representing the sequence, and location, of RNA corresponding to every gene in the human genome.
Lee, Daugharthy and their colleagues then tested the method to detect the genes skin cells turn on as they multiply and migrate to heal a simulated wound in a petri dish. Cells growing into the wound had 12 genes that were activated much more or much less than nearby cells sitting idly on the sidelines. Similar experiments could identify new markers of diseased tissue or new targets for targeted molecular therapies.
“What George’s team has accomplished is a technological tour de force,” said Wyss Institute founding director Don Ingber. “By spotting incredibly subtle but incredibly important changes in gene expression and precisely defining their position inside the cell, they have helped open the door to a new age of cellular diagnostics.”
The work was funded by the National Institutes of Health, the Allen Institute for Brain Science and the Wyss Institute.
A new microscopy method could enable scientists to generate snapshots of dozens of different biomolecules at once in a single human cell, a team from the Wyss Institute of Biologically Inspired Engineering at Harvard University reported Sunday in Nature Methods.
Such images could shed light on complex cellular pathways and potentially lead to new ways to diagnose disease, track its prognosis, or monitor the effectiveness of therapies at a cellular level.
Cells often employ dozens or even hundreds of different proteins and RNA molecules to get a complex job done. As a result, cellular job sites can resemble a busy construction site, with many different types of these tiny cellular workers coming and going. Today's methods typically only spot at most three or four types of these tiny workers simultaneously. But to truly understand complex cellular functions, it's important to be able to visualize most or all of those workers at once, said Peng Yin, assistant professor of systems biology at Harvard Medical School and a core faculty member at the Wyss Institute.
"If you can see only a few things at a time, you are missing the big picture," Yin said.
Yin's team sought a way to take aerial views of job sites that could spot up to dozens of types of biomolecules that make up large cellular work crews.
To capture ultrasharp images of biomolecules, they had to overcome laws of physics that stymied microscopists for most of the last century. When two objects are closer than about 200 nanometers apart — about one five-hundredth the width of a human hair — they cannot be distinguished using a traditional light microscope: the viewer sees one blurry blob where in reality there are two objects.
Since the mid-1990s, scientists have developed several ways to overcome this problem using combinations of specialized optics, special fluorescent proteins or dyes that tag cellular components.
Ralf Jungmann, now a postdoctoral Fellow working with Yin at the Wyss Institute and Harvard Medical School, helped develop one of those super-resolution methods, called DNA-PAINT, as a graduate student. DNA-PAINT can create ultrasharp snapshots of up to three cellular workers at once by labeling them with different colored dyes.
To visualize cellular job sites with crews of dozens of cellular workers, Yin's team, including Jungmann, Maier Avendano, M.S., a graduate student at Harvard Medical School, and Johannes Woehrstein, a postgraduate research fellow at the Wyss Institute, modified DNA-PAINT to create a new method called Exchange-PAINT.
Exchange-PAINT relies on the fact that DNA strands with the correct sequence of letters, or nucleotides, bind specifically to partner strands with complementary sequences. The researchers label a biomolecule they want to visualize with a short DNA tag, then add to the solution a partner strand carrying a fluorescent dye that lights up only when the two strands pair up. When that partner strand binds the tagged biomolecule, it lights up, then lets go, causing the biomolecule to "blink" at a precise rate the researchers can control. The researchers use this blinking to obtain ultrasharp images.
They then repeat the process to visualize a second target, a third, and so on. Then they overlay the resulting images to create a composite image in which each biomolecule — each cellular worker — is assigned a different color. This allows them to create false-color images that simultaneously show many types of biomolecules — far more than they could simultaneously visualize by labeling them with different colored dyes. And these false-color images allow them to spot enough cellular workers at once to capture the entire scene.
To test Exchange-PAINT, the researchers created 10 unique pieces of folded DNA, or DNA origami, that resembled the numerals 0 through 9. These numerals could be resolved with less than 10 nanometers resolution, or one-twentieth of the diffraction limit.
The team was able to use Exchange-PAINT to capture clear images of the 10 different types of miniscule DNA origami structures in one image. They also used the method to capture detailed, ultrasharp images of fixed human cells, with each color tagging an important cellular component — microtubules, mitochondria, Golgi apparatus, or peroxisomes.
Yin expects the method, with further development, to be able to visualize dozens of cellular components at once.
"Peng's exciting new imaging work gives biologists an important new tool to understand how multiple cellular components work together in complex pathways," said Wyss Institute Founding Director Don Ingber. "I expect insights from those experiments to lead to new ways to diagnose and monitor disease." Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital and Professor of Bioengineering at Harvard School of Engineering & Applied Sciences.
In addition to Yin, Jungmann, Avendano, and Woehrstein, the team included Mingjie Dai, a graduate student in biophysics at Harvard University and William Shih, a Wyss Institute core faculty member who is also associate professor of biological chemistry and molecular pharmacology at Harvard Medical School and associate professor of cancer biology at the Dana-Farber Cancer Institute. The work was funded by the National Institutes of Health, the Office of Naval Research, the National Science Foundation, the Humboldt Foundation, and the Wyss Institute.
Adapted from a Wyss Institute news release.
Sharpening CRISPR-Cas’s Aim
A simple adjustment to a powerful gene-editing tool improves its precision, Harvard Medical School researchers at Massachusetts General Hospital report.
In a paper published in Nature Biotechnology, the scientists have shown how adjusting the length of guide RNAs in synthetic enzymes called CRISPR-Cas RNA-guided nucleases can substantially reduce off-target DNA mutations, a limitation the team revealed just last year.
“Simply by shortening the length of the guide RNA targeting region, we saw reductions in the frequencies of unwanted mutations at all of the previously known off-target sites we examined,” said J. Keith Joung, HMS associate professor of pathology at Mass General and senior author of the paper. “Some sites showed decreases in mutation frequency of 5,000-fold or more, compared with full-length guide RNAs. Importantly, these truncated guide RNAs—which we call tru-gRNAs—are just as efficient as full-length gRNAs at reaching their intended target DNA segments.”
Last year two groups reported their success in using a tool borrowed from a bacterial immune system called Cas, short for CRISPR-associated systems, which in turn stands for Clustered Regularly Interspaced Short Palindromic Repeats. In bacteria the Cas9 enzyme system uses short stretches of RNA to target and then cut invading viral DNA. Scientists have customized this system to work in human cells, creating an RNA-guided editing tool that allows them to integrate DNA changes into the genomes of living cells.
Later last year Joung’s team found that in human cells, CRISPR-Cas RNA-guided nucleases could also cause mutations in DNA sequences with differences of up to five nucleotides from the target, which could seriously limit the proteins’ clinical usefulness. The team followed up with a hypothesis that could seem counterintuitive: Shortening the gRNA segment might reduce off-target mutations.
“Some of our experiments from last year suggested that one could mismatch a few nucleotides at one end of the gRNA complementarity region without affecting the targeting activity,” Joung explained. “That led us to wonder whether removing these nucleotides could make the system more sensitive to mismatches in the remaining sequence.”
The CRISPR-Cas RNA-guided nucleases most widely used by researchers include a 20-nucleotide targeting region within the gRNA. To test their theory, the team constructed RNA-guided nucleases with progressively shorter gRNAs. They found that while gRNAs with targeting segments of 17 or 18 nucleotides were at least as efficient as full-length gRNAs in reaching their targets, those with 15- or 16-nucleotide targeting segments had reduced or no targeting activity. Subsequent experiments found that 17-nucleotide truncated RNA-guided nucleases efficiently induced the desired mutations in human cells, with greatly reduced or undetectable off-target effects, even at sites with only one or two mismatches.
“While we don’t fully understand the mechanism by which tru-gRNAs reduce off-target effects, our hypothesis is that the original system might have more energy than it needs, enabling it to cleave even imperfectly matched sites,” Joung said. “By shortening the gRNA, we may reduce the energy to a level just sufficient for on-target activity, making the nuclease less able to cleave off-target sites. But more work is needed to define exactly why tru-gRNAs have reduced off-target effects.”
Joung’s team has incorporated this capability for finding tru-gRNA target sites into ZiFiT Targeter, a freely available software package designed to identify potential target sites for several DNA-editing technologies.
The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM088040 and P50 HG005550, and the Jim and Ann Orr MGH Research Scholar Award.
Adapted from a Mass General news release.
A new technology developed by Harvard Medical School researchers at the Massachusetts General Hospital Center for Systems Biology allows the simultaneous analysis of hundreds of cancer-related protein markers from minuscule patient samples gathered through minimally invasive methods. This powerful and sensitive technology uses antibodies linked to unique DNA “barcodes” to detect a wide range of target proteins.
It could serve as a tool to help clinicians gain insights into the biology of cancer progression as well as determine why certain cancer therapies stop working or are ineffective to begin with. Development of the technology is reported in Science Translational Medicine.
Minimally invasive techniques—such as fine-needle aspiration or circulating tumor cell analysis—are increasingly employed to track treatment response over time in clinical trials, as the tests can be simple and cheap to perform. Fine-needle aspirates are also much less invasive than core biopsies or surgical biopsies, since very small needles are used. The challenge has been to comprehensively analyze the very few cells that are obtained via this method.
“What this study sought to achieve was to vastly expand the information that we can obtain from just a few cells,” explained Cesar Castro, HMS instructor in medicine at Mass General and a co-author of the paper. “Instead of trying to procure more tissue to study, we shrank the analysis process so that it could now be performed on a few cells.”
Until now, pathologists have been able to examine only a handful of protein markers at a time for tumor analyses. With this new technology, the researchers have demonstrated the ability to look at hundreds of markers simultaneously, down to the single-cell level.
“We are no longer limited by the scant cell quantities procured through minimally invasive procedures,” Castro said. “Rather, the bottleneck will now be our own understanding of the various pathways involved in disease progression and drug target modulation.”
The new method uses an approach known as DNA-barcoded antibody sensing, in which unique DNA sequences are attached to antibodies against known cancer marker proteins. The DNA “barcodes” are linked to the antibodies with a special type of glue that breaks apart when exposed to light. When mixed with a tumor sample, the antibodies seek out and bind to their targets; then a light pulse releases the unique DNA barcodes of these bound antibodies that are subsequently tagged with fluorescently labeled complementary barcodes. The tagged barcodes can be detected and quantified via imaging, revealing which markers are present in the sample.
After initially demonstrating and validating the technique’s feasibility in cell lines and single cells, the team tested it on samples from patients with lung cancer. The technology was able to reflect the great heterogeneity—differences in features such as cell-surface protein expression—of cells within a single tumor and to reveal significant differences in protein expression between tumors that appeared identical under the microscope. Examination of cells taken at various time points from participants in a clinical trial of a targeted therapy drug revealed patterns that distinguished those who did and did not respond to treatment.
“We showed that this technology works well beyond the highly regulated laboratory environment, extending into early-phase clinical trials,” said Castro, who is also a medical oncologist in the Mass General Cancer Center and director of the Cancer Program within the hospital’s Center for Systems Biology. “In this era of personalized medicine, we could leverage such technology not only to monitor but actually to predict treatment response. By obtaining samples from patients before initiating therapy and then exposing them to different chemotherapeutics or targeted therapies, we could select the most appropriate therapy for individual patients.”
Ralph Weissleder, the HMS Thrall Family Professor of Radiology at Mass General, is corresponding author of the Science Translational Medicine paper. The study was funded in part by National Cancer Institute grant U54 CA15188.
Adapted from a Mass General news release.
A gene mutation associated with several types of cancer may also be responsible for a rare but debilitating brain tumor called papillary craniopharyngioma, according to a team led by HMS investigators from Massachusetts General Hospital, Brigham and Women’s Hospital, Dana-Farber Cancer Institute and the Broad Institute. The discovery, reported in Nature Genetics, could lead to new therapies for this currently hard-to-treat tumor.
“We were delighted to find that the same BRAF mutation previously described in melanomas and other brain tumors appears to be driving the growth of these tumors,” said Priscilla Brastianos, HMS instructor in medicine at Mass General, a research associate at the Broad Institute and co-corresponding author of the paper. “BRAF inhibitors have shown great promise in treating patients with other tumors with this mutation, and we hope to quickly evaluate these drugs in patients with papillary craniopharyngioma in hopes of reducing the serious consequences of this disease.”
Craniopharyngiomas arise at the base of the skull adjacent to the pituitary gland, the hypothalamus and other critical brain structures. Although they are not inherently aggressive tumors, their location means they can significantly compromise vision and other neurologic and endocrine functions. The tumors cling to brain structures, making surgical removal challenging. Radiation therapy can cause vascular abnormalities or other tumors.
There are two subtypes of craniopharyngiomas: adamantinomatous tumors, which are more common in children, and papillary tumors, usually seen in adults. Recent studies have linked mutations in the cancer-causing gene CTNNB1 with adamantinomatous tumors, but before this study, no information was available about the molecular drivers of the papillary subtype.
In their search for possible mutations associated with papillary tumors, the research team first performed whole exome sequencing of 12 adamantinomatous and three papillary craniopharyngiomas. The CTNNB1 mutation was found in 11 of the 12 adamantinomatous samples. For the first time, the well-known tumor-causing BRAF mutation was identified in all three papillary tumors.
The researchers followed that finding with targeted genotyping of tumor samples from an additional 95 patients. Ninety-four percent of the papillary tumors they tested carried the BRAF mutation, while 96 percent of the adamantinomatous tumors had the CTNNB1 mutation. The investigators also confirmed that both types of tumors had very few other mutations. The BRAF or CTNNB1 mutations were present in all tumor cells, suggesting they occurred early in tumor development.
“There are currently no medical therapies available for craniopharyngiomas, but potent compounds that block BRAF signaling are in hand. So we are very hopeful that these targeted therapies can drastically alter the management of these tumors,” said Sandro Santagata, HMS assistant professor of pathology at Brigham and Women’s and a co-corresponding author of the paper. “Inhibitors of the signaling pathway controlled by CTNNB1 that are currently in clinical trials should be investigated for adamantinomatous tumors, and we’re planning to evaluate the BRAF inhibitors that have had promising results against melanoma for treatment of papillary craniopharyngiomas.”
Along with Brastianos, the co-lead authors of the report are Amaro Taylor-Weiner of the Broad Institute and Peter Manley, HMS instructor in pediatrics at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center. Co-senior authors are Mark Kieran, HMS associate professor of pediatrics at Dana-Farber/Boston Children’s, and Gad Getz, HMS associate professor of pathology at Mass General and the Broad Institute. David Louis, the Benjamin Castleman Professor of Pathology at Mass General, also made significant contributions to the study, as did additional collaborators from Mass General, Brigham and Women’s, Dana-Farber, Johns Hopkins University, the University of Pennsylvania and other institutions.
Support for the study includes grants from Pedals for Pediatrics and the Jared Branfman Sunflowers for Life Foundation for Pediatric Brain and Spinal Cord Research.
Adapted from a Mass General news release.
Cervical Cancer Clues
Researchers from the Boston area, Mexico and Norway have completed a comprehensive genomic analysis of cervical cancer in two patient populations. The study identified recurrent genetic mutations not previously found in cervical cancer, including at least one for which targeted treatments have been approved for other forms of cancer. The findings also shed light on the role human papillomavirus (HPV) plays in the development of cervical cancer.
The study, which appears online in Nature, addresses a public health concern of global significance. Cervical cancer is the second most common cancer in women and is responsible for approximately 10 percent of cancer deaths in women—particularly in developing countries where screening methods are not readily accessible. Almost all cases of the disease are caused by exposure to HPV and it is expected that vaccination efforts targeting HPV will decrease cervical cancer cases over time. In the meantime, however, the disease remains a significant threat to women’s health.
“Cancer is a disease that affects the whole world, and one question always arises: Is a given cancer type similar or different across populations?” explained Matthew Meyerson, one of the paper’s co-senior authors. Meyerson is an HMS professor of pathology and medical oncology at Dana-Farber Cancer Institute and a senior associate member of the Broad Institute. “While we don’t have the complete answer yet in this case, what we are seeing is that, in two different populations, the causes of cervical cancer are similar and, fundamentally, in both cases it comes down to HPV-genome interaction.”
To investigate the genomic underpinnings of the disease, the team performed whole-exome sequencing, which examines the genetic code in the protein-coding regions of the genome, on samples from 115 cervical cancer patients from Norway and Mexico. In some cases, the researchers also conducted whole-genome sequencing (analyzing the genetic code across the entire genome) or transcriptome sequencing (focusing on gene expression). In each case, the researchers compared genomic data derived from cervical cancer tumors with genomic data from healthy tissue from the same individual to determine what may have gone wrong—or mutated—in the genome to allow the cancer to develop. The mutations identified in tumors but not in healthy tissues from the same individuals are referred to as somatic mutations.
The study benefited from the international collaboration of scientists from research institutes across the globe and was made possible by the Slim Initiative for Genomic Medicine in the Americas (SIGMA), which promotes the study of genomic medicine in the service of global health.
“Low- and middle-income countries suffer the largest burden of cancer in the world,” said co-author Jorge Melendez of the National Institute of Genomic Medicine in Mexico City. “Nevertheless, only 5 percent of all the global resources dedicated to this group of diseases are allocated to them. Initiatives that promote joint efforts with developing countries will help to advance not only the knowledge of the shared and distinct biological aspects of cancer diseases, but also highlight local action items to impact public health.”
The cooperation of teams from the U.S., Mexico and Norway was essential in order to sequence samples from a diverse pool of cervical cancer patients.
“Without this sort of international collaboration, the genomic view of a disease can be limited. By analyzing genomic data from diverse populations, we can discover patterns of disease progression in context of the full range of human genetic variation,” said co-senior author Helga Salvesen. Salvesen, a professor of clinical medicine at the University of Bergen, Norway, was a visiting scientist at the Broad Institute when the study was conducted.
The study identified 13 mutations that occurred frequently enough across the samples to be considered significant in cervical cancer. Eight of these mutations had not been linked to the disease previously, and two had not previously been seen in any cancer type.
Among the most notable findings were somatic point mutations in the gene ERBB2, which was found in a small but significant subset of the tumors. Mutations in this gene, which is also known as HER-2, had not been previously linked to cervical cancer, but it is a known oncogene common in breast cancer. Treatments exist that target the gene.
“This suggests that a subset of cervical cancer patients could be candidates for clinical trials involving ERBB2 inhibitors, which are available and FDA approved,” explained Akinyemi Ojesina, an HMS postdoctoral fellow in Meyerson’s lab at the Broad Institute and Dana-Farber. Ojesina served as a co-first author of the paper along with Lee Lichtenstein of the Broad’s Cancer Genome Analysis Group. “It is an exciting finding that could be translatable to the clinic.”
The team also identified a novel mutation in the gene MAPK1. MAPK1 is one of the final steps in the MAP kinase signaling pathway—a network of interconnected genes that play a role in cell growth regulation. Mutations in other genes in the pathway have been known to drive cancer, but this is the first time that MAPK1 itself has been found to be mutated. The finding opens up the possibility that MAPK1, like other genes in the MAP kinase signaling pathway, may be a viable therapeutic target.
Another key finding was the prevalence of mutations in genes affecting the immune system, in particular those in the gene HLA-A, which helps the body distinguish its own proteins from foreign invaders. Mutations in HLA-A were previously found to drive squamous cell lung cancer. In this study, another gene in the same complex—HLA-B—was found to be commonly mutated in cervical squamous cell carcinoma. This suggests that disruptions to the immune system may play a bigger role in cancer progression than was previously realized.
Finally, transcriptome sequencing, which allowed the team to analyze gene expression—how and when genes are activated across the genome—enabled the researchers to learn more about how HPV is driving cervical cancer.
It has long been known that exposure to HPV is a primary risk factor for cervical cancer. Once an individual is exposed, the immune system often clears out the infection, but in cases in which the virus lingers, it can integrate itself into the human genome. This study looked at where in the genome HPV inserted itself. The scientists found that HPV integration sites were associated with higher levels of gene expression and were often amplified, resulting in many copies of those sections of the genome. This connection between HPV integration and gene expression suggests that the virus may be driving cancer by promoting and elevating the activity of mutated genes.
“Our findings further elucidate the key role HPV is playing in the development of cervical cancer, which in turn emphasizes the importance of combating the disease by vaccinating against HPV,” Meyerson said.
In addition to the evidence supporting vaccination as a means of prevention, the researchers say the study bears important clinical implications for targeted therapeutics.
“In metastatic cervical cancer, more effective systemic therapy is urgently needed,” Salvesen explained. “So far, our knowledge regarding genetic alterations as potential targets for therapeutics has been limited, and no targeted therapeutics are yet in routine clinical use. The present study—in particular the findings related to ERBB2—thus represents a unique and comprehensive new tool to guide clinical trial design in the future.”
“The outstanding findings of our successful collaborative international research is giving us, here in Mexico, very powerful arguments in favor of the benefit of speeding up the adoption of molecular methods for screening HPV, followed by colposcopy to detect cancer at its earliest phases when it is curable; not to mention the possibility of new targeted therapies that our discoveries open up,” said Hugo Barrera, a professor of biochemistry and molecular medicine at the School of Medicine and University Hospital of the University of Nuevo Leon in Monterrey, Mexico, who, with Melendez, coordinated the Mexican team.
“It is hoped that, as we combine vaccination strategies and novel targeted therapies, we will be better able to combat the scourge that is cervical cancer,” Ojesina added.
This work was conducted as part of the Slim Initiative for Genomic Medicine in the Americas, a project funded by the Carlos Slim Foundation through the Health Institute. This work was also partially supported by the Rebecca Ridley Kry Fellowship of the Damon Runyon Cancer Research Foundation; MMRF Research Fellow Award; Helse Vest, Research Council of Norway, Norwegian Cancer Society and Harald Andersens legat; CONACyT grant SALUD-2008-C01-87625 and UANL PAICyT grant CS1038-1; and CONACyT grant 161619.
Adapted from a Broad Institute news release.
A New—and Reversible—Cause of Aging
Researchers have discovered a cause of aging in mammals that may be reversible.
The essence of this finding is a series of molecular events that enable communication inside cells between the nucleus and mitochondria. As communication breaks down, aging accelerates. By administering a molecule naturally produced by the human body, scientists restored the communication network in older mice. Subsequent tissue samples showed key biological hallmarks that were comparable to those of much younger animals.
“The aging process we discovered is like a married couple—when they are young, they communicate well, but over time, living in close quarters for many years, communication breaks down,” said Harvard Medical School Professor of Genetics David Sinclair, senior author on the study. “And just like with a couple, restoring communication solved the problem.”
This study was a joint project between Harvard Medical School, the National Institute on Aging, and the University of New South Wales, Sydney, Australia, where Sinclair also holds a position.
The findings are published Dec. 19 in Cell.
Mitochondria are often referred to as the cell's "powerhouse," generating chemical energy to carry out essential biological functions. These self-contained organelles, which live inside our cells and house their own small genomes, have long been identified as key biological players in aging. As they become increasingly dysfunctional overtime, many age-related conditions such as Alzheimer’s disease and diabetes gradually set in.
Researchers have generally been skeptical of the idea that aging can be reversed, due mainly to the prevailing theory that age-related ills are the result of mutations in mitochondrial DNA—and mutations cannot be reversed.
Sinclair and his group have been studying the fundamental science of aging—which is broadly defined as the gradual decline in function with time—for many years, primarily focusing on a group of genes called sirtuins. Previous studies from his lab showed that one of these genes, SIRT1, was activated by the compound resveratrol, which is found in grapes, red wine and certain nuts.
Ana Gomes, a postdoctoral scientist in the Sinclair lab, had been studying mice in which this SIRT1 gene had been removed. While they accurately predicted that these mice would show signs of aging, including mitochondrial dysfunction, the researchers were surprised to find that most mitochondrial proteins coming from the cell’s nucleus were at normal levels; only those encoded by the mitochondrial genome were reduced.
“This was at odds with what the literature suggested,” said Gomes.
As Gomes and her colleagues investigated potential causes for this, they discovered an intricate cascade of events that begins with a chemical called NAD and concludes with a key molecule that shuttles information and coordinates activities between the cell’s nuclear genome and the mitochondrial genome. Cells stay healthy as long as coordination between the genomes remains fluid. SIRT1’s role is intermediary, akin to a security guard; it assures that a meddlesome molecule called HIF-1 does not interfere with communication.
For reasons still unclear, as we age, levels of the initial chemical NAD decline. Without sufficient NAD, SIRT1 loses its ability to keep tabs on HIF-1. Levels of HIF-1 escalate and begin wreaking havoc on the otherwise smooth cross-genome communication. Over time, the research team found, this loss of communication reduces the cell's ability to make energy, and signs of aging and disease become apparent.
“This particular component of the aging process had never before been described,” said Gomes.
While the breakdown of this process causes a rapid decline in mitochondrial function, other signs of aging take longer to occur. Gomes found that by administering an endogenous compound that cells transform into NAD, she could repair the broken network and rapidly restore communication and mitochondrial function. If the compound was given early enough—prior to excessive mutation accumulation—within days, some aspects of the aging process could be reversed.
Examining muscle from two-year-old mice that had been given the NAD-producing compound for just one week, the researchers looked for indicators of insulin resistance, inflammation and muscle wasting. In all three instances, tissue from the mice resembled that of six-month-old mice. In human years, this would be like a 60-year-old converting to a 20-year-old in these specific areas.
One particularly important aspect of this finding involvesHIF-1. More than just an intrusive molecule that foils communication, HIF-1 normally switches on when the body is deprived of oxygen. Otherwise, it remains silent. Cancer, however, is known to activate and hijack HIF-1. Researchers have been investigating the precise role HIF-1 plays in cancer growth.
“It’s certainly significant to find that a molecule that switches on in many cancers also switches on during aging,” said Gomes. “We're starting to see now that the physiology of cancer is in certain ways similar to the physiology of aging. Perhaps this can explain why the greatest risk of cancer is age.”
“There’s clearly much more work to be done here, but if these results stand, then certain aspects of aging may be reversible if caught early,” said Sinclair.
The researchers are now looking at the longer-term outcomes of the NAD-producing compound in mice and how it affects the mouse as a whole. They are also exploring whether the compound can be used to safely treat rare mitochondrial diseases or more common diseases such as Type 1 and Type 2 diabetes. Longer term, Sinclair plans to test if the compound will give mice a healthier, longer life.
The Sinclair lab is funded by the National Institute on Aging (NIA/NIH), the Glenn Foundation for Medical Research, the Juvenile Diabetes Research Foundation, the United Mitochondrial Disease Foundation and a gift from the Schulak family.