Stephen J. Elledge Receives Lasker Award

Geneticist recognized for discovering DNA repair pathway

Stephen J. Elledge, the Gregor Mendel Professor of Genetics and of Medicine at HMS, and professor of medicine at Brigham and Women’s Hospital, is a co-recipient, with Evelyn Witkin of Rutgers University, of the 2015 Albert Lasker Basic Medical Research Award.

The award, widely considered to be among the most respected in biomedicine, will be presented on Friday, Sept. 18, in New York City.

Elledge and Witkin are being honored for their seminal discoveries that have illuminated the DNA damage response, a cellular pathway that senses when DNA is altered and sets in a motion a series of responses to protect the cell. This pathway is critical to a better understanding of many diseases and conditions, such as cancer.

"Steve is an amazing scientist, mentor and colleague," said Jeffrey S. Flier, dean of Harvard Medical School.

"His insights into the basic mechanisms of the DNA damage response have profoundly enriched our understanding not only of the fundamental genetics of all cellular life, but also of how we conceptualize many diseases and conditions, especially cancer. This distinction is richly deserved, and I am delighted that Steve is being honored for this extraordinary body of work," Flier said.

"We are extremely proud of Steve, who is truly deserving of this recognition,” said Elizabeth G. Nabel, MD, president of Brigham and Women's Health Care.

“Courageous and insatiably inquisitive, he represents the best of Brigham and Women’s and our mission of driving innovation in basic science to improve human health. As a devoted mentor, Steve is deeply committed to guiding the careers of young investigators, ensuring that the next generation of scientists is filled with curious, passionate and talented researchers," she added.

Elledge often describes the process by which a cell duplicates itself as akin to the duplication of a small city. It is a vastly complex process that requires many levels of intricate coordination. Each cell contains a detailed blueprint for this entire process: DNA.

But not every duplication results in a perfect copy. That is because each time a cell makes a copy of itself, DNA is vulnerable to damage, not only from faulty cellular processes, but also from such entities as environmental chemicals. As DNA damage accumulates, it profoundly complicates a cell’s ability to make a faithful copy of itself. This can lead to serious illnesses, birth defects, cancer and other health problems.

Witkin discovered how bacteria respond to DNA damage, detailing the response to UV radiation. Elledge uncovered a DNA-damage response pathway that operates in more complex organisms, including humans.

Over the years, Elledge and his colleagues elucidated a signaling network that informs a cell when DNA sustains an injury.

Called the DNA damage response, this network senses the problem and sends a signal to the rest of the cell so it can properly repair itself, otherwise severe mutations can occur. As a result, this pathway helps keep the genome stable and suppresses adverse events such as tumor development.

When individuals are born with mutations in this pathway, they often have severe developmental defects. If the pathway is interfered with later in life, cancer can result.

In addition to the award in basic medical research, the Lasker Foundation is also presenting awards to individuals in clinical research and in public service.

According to Claire Pomeroy, president of the Albert and Mary Lasker Foundation, this year’s recipients “remind us all that investing in biological sciences and medical research is crucial for our future.”

Joseph L. Goldstein of the University of Texas Southwestern Medical Center and chair of the Lasker Medical Research Awards Jury, added, “The 2015 Lasker winners had bold ideas and pursued novel questions that they tested through fearless experimentation.”


Damage Control

Researchers connect pathway linked to Parkinson’s disease with genes mutated in ALS

When mitochondria—the cell’s power plant—are sick or damaged, they must be cleared away so the cell can survive. 

In the brain, this mitochondrial quality control pathway is so critical that neurodegenerative disease can result if bad mitochondria accumulate in neurons.

Now, Harvard Medical School researchers have mechanistically connected this pathway—which is already linked to Parkinson’s disease—with proteins that are mutated in amyotrophic lateral sclerosis, the motor-neuron disease also known as Lou Gehrig’s disease. Their findings are described in Molecular Cell.

In healthy cells, damaged mitochondria are broken down and disposed of by a process called selective autophagy, a term stemming from Greek roots that translates as “self-eating.” How the mitochondria get tagged for disposal has been the subject of research in many labs around the world in recent years.

Proteins linked to Parkinson’s

Mitochondrial damage triggers activation of two proteins—PARKIN and PINK1—that tag the mitochondria surface with chains of ubiquitin, molecules that signal the cell to get rid of the defective mitochondria. These proteins have been known for more than a decade to be faulty in early-onset familial forms of Parkinson’s disease, but only recently have their roles in mitochondrial quality control been elucidated.

Most recently, scientists have been studying how cells recognize these disposal signals and which proteins are involved further downstream in the disposal process.

Initial key insights into the downstream mechanism were suggested several years ago by the finding that the autophagy cargo receptor protein OPTN functions together with the TBK1 protein kinase in the removal of pathogenic bacteria from cells via autophagy. Interestingly, both OPTN and very recently TBK1 were also found to be mutated in ALS, but how these proteins contribute to this neurodegenerative disease has remained poorly understood.

Now scientists led by Wade Harper have mechanistically connected these two sets of proteins and have described a multistep mitochondrial quality control pathway. Upstream proteins—PARKIN and PINK1—function early in the mitochondrial disposal process by assembling ubiquitin chains on damaged mitochondria while the downstream proteins—OPTN and TBK1—bind to these ubiquitin chains to target the damaged mitochondria to the autophagy machinery.

Importantly, binding of the OPTN-TBK1 complex to these ubiquitin chains promotes TBK1 activation and further activation of OPTN’s ubiquitin-binding activity, establishing a self-reinforced feed-forward mechanism that is critical for the ultimate delivery of mitochondria to the autophagosome.

Connection to ALS

By using state-of-the-art quantitative mass spectrometry coupled with gene-editing and cell-imaging tools, the new study provides a detailed picture of how these two pathways work together and suggests how they may link the two neurodegenerative diseases.

“This is the first time we could mechanistically show how ubiquitination of damaged mitochondria promotes their clearance, and suggests that mitophagy defects potentially contribute to ALS as well as Parkinson’s disease,” said Harper, who is Bert and Natalie Vallee Professor of Molecular Pathology and chair of the HMS Department of Cell Biology.

Alban Ordureau, a postdoctoral fellow in the Harper lab and a co-author of the paper, had previously studied how PARKIN and PINK1 work together biochemically to specifically tag damaged mitochondria with ubiquitin.

“The story here focuses on how, once mitochondria are ubiquitinated, they are recognized by the cell and gotten rid of by proteins implicated in two distinct neurodegenerative diseases,” said Ordureau.

Jin-Mi Heo, postdoctoral fellow in the Harper lab and lead author of the paper, discovered that TBK1 becomes activated when mitochondria are damaged via ubiquitin chain binding by OPTN.

Stress and sensitivity

“If there is stress happening in the same cell in one pathway, that might cause defects in other pathways,” Heo said. “That could be why we see similar characteristics in different diseases with different origins.”

In Parkinson’s disease, neurons that make the neurotransmitter dopamine are defective while in ALS, the motor neurons do not perform as they should.

“There are some very complex relationships between different mutations that are going to lead to disease and which cell types they act in,” Harper said. “Depending on the cell type, you get one or the other disease.”

One idea is that individual types of neurons are sensitive to toxic proteins or toxic organelles—such as damaged mitochondria—in different ways. Motor neurons, for example, are much longer than most neurons in the brain, so they may be more sensitive to the accumulation of certain types of toxic proteins or organelles than other types of neurons, Harper said. The mutations that are present in the individual diseases may reflect sensitivities to different types of autophagic cargo; the result is motor neuron disease in ALS or dopaminergic disease in Parkinson’s.

“The surprising thing is that the Parkinson’s genes are functioning upstream of a pathway that’s mutated downstream in motor neuron disease,” Harper said. “So there is a genetic sensitivity within the pathway that must be different in different cells.”

It may turn out that this is a general mechanism that cells use to get rid of a variety of damaged material in different kinds of neurons.

“We’re starting to work that out,” Harper said.

This study was supported by National Institutes of Health grant R37 NS083524, Biogen and the Edward R. and Anne G. Lefler Center for the Study of Neurodegenerative Disorders at HMS.  Harper is a consultant for Millennium: the Takeda Oncology Company and Biogen.


Global Ambition

Cancer biology and therapeutics program debuts 

Harvard Medical School and the Qatar Biomedical Research Institute, with a commitment from the Shenzhen-HMS Initiative in International Education in China, are working together to make history with a joint global initiative designed to disseminate knowledge of cancer biology, prevention and treatment.

A new 12-month program aimed at international clinicians and scientists who specialize in cancer, Cancer Biology and Therapeutics will break new ground as the first HMS global program to combine basic and applied science in a blended-learning format, providing insights from leading researchers, oncologists and therapeutic developers both online and in person.

“This collaborative initiative will benefit Qatar Biomedical Research Institute, the Shenzhen-HMS Initiative in International Education and Harvard Medical School through the exchange of ideas and will develop a regional and global network of future leaders in cancer research and treatment,” said Ajay Singh, HMS associate dean for global education and continuing education.

HMS-CBT, as the program is known, highlights the global nature of the HMS mission, said George Demetri, co-director of the Harvard Ludwig Center, HMS professor of medicine at Dana-Farber Cancer Institute and a co-director of HMS-CBT.

The deadline for applications is Oct. 14.

“We are very focused on the terrific talent we’re pleased to have in the United States, but many brilliant people outside the United States are not able to come here for their training,” Demetri said.

“The more we can share the expanding body of knowledge about cancer biology and therapeutics with them, I think the better it will be for patients and for the field,” he said.

Combining the study of cancer biology with the study of cancer therapeutics is intentional and necessary, Demetri said.

“We have had an explosion in knowledge relating to cancer—the diversity of cancers, the fact that cancer is not one disease but probably thousands of different diseases that we can now parse very finely in the new world of precision cancer medicine,” he said.

“How do we develop therapeutics from that knowledge? How do we apply those therapeutics? That has so much depth and robustness we can address in this program,” Demetri added.

Emphasis on interactivity

Through a combination of three in-person, three-day workshops in cities on three continents—in Doha (Qatar), London and Boston—the program emphasizes interactivity between students and Harvard faculty and between groups of students who will organize themselves via internet-connected teams around projects and ideas.

The program, which begins Oct. 24 with a workshop in Doha, will be multi-faceted, reflecting both the evolving science of cancer and the interests of its students.  The first workshop will feature an introductory session on cancer biology, followed by sessions focused on links between infectious diseases, viruses and cancer.

The second workshop, starting March 18, 2016, in London, will focus on targeted therapies in cancer.

The third workshop and capstone event for the program, beginning Oct. 26, 2016, in Boston, will feature the latest developments in cancer immunotherapy, a dynamic field that is changing so rapidly that the organizers predict topics will evolve over the coming year.

Students will also be asked about what they are particularly interested in learning.


HMS-CBT program co-director Ed Harlow, the Virginia and D.K. Ludwig Professor of Cancer Research and Teaching in the HMS Department of Biological Chemistry and Molecular Pharmacology, is well known for Provocative Questions, a program he initiated in 2012 at the National Cancer Institute with former director Harold Varmus.  Harlow remains special assistant to the current NCI director.

Harlow hopes to spark the same impulse with students at the first HMS-CBT workshop by inviting them to ask questions for which there may not be one simple answer.

“This sounds interesting to me, this idea that an international group of scientists will be talking to one another about this project they’ve got to work on, and how they’ll do it across global time zones and cultures,” Harlow said.

Demetri calls this adaptable form of education “bespoke.” While a library of lectures from Harvard faculty is being assembled, webinars and interaction among students and with professors will supplement them, addressing the needs and interests of the students as they emerge. 

Peer-to-peer projects

The program directors are committed to interactivity, which they say will sprout at the workshops and flower during the year through peer-to-peer projects.

“The course will create opportunities for the students to network,” said Peter Howley, the Shattuck Professor of Pathological Anatomy in the HMS Department of Microbiology and Immunobiology and an HMS-CBT co-director.

“One could imagine wonderful and creative outcomes from such learning collaborations. The course faculty will give them assignments and will be around to help the students refine and work on them, but the actual learning is going to be among themselves,” he said.

In the process, the program directors will actively assess the course to learn what succeeds, and, along with the students, will work to build upon the experiences for future offerings of this course.

”We want to provide an educational experience that’s useful for all the participants,” Demetri said.

“It is really going to be an exciting year.”   


Fat in the Family?

Study could lead to therapeutics that boost metabolism

Like many other conditions, obesity is caused by an interplay between genetic and environmental factors. While efforts to combat the obesity epidemic will need to include changes in diet and exercise, insights into the genes involved may also help with prevention and treatment.

Now a research team led by Harvard Medical School investigators at Beth Israel Deaconess Medical Center and MIT reveals the mechanistic explanation behind the strongest genetic association with obesity.

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The findings, published in the New England Journal of Medicine, uncover a genetic circuit that controls whether our bodies burn or store fat. Manipulating that genetic circuit may offer a new approach for obesity treatments.

The strongest genetic association with obesity lies within an unexpressed region of the FTO gene and contains 89 common variants in a region of 47,000 nucelotides.

The obesity-risk version of the region that predisposes individuals to increased body weight is found in 44 percent of individuals in European populations, but its mechanistic basis has until now remained unknown, despite extensive investigation.

Assessing epigenetic modifications

To identify the cell types in which the FTO obesity-risk region might exert its effects, the researchers analyzed information from the Roadmap Epigenomics project, which assesses chemical or “epigenetic” modifications within chromosomes that switch genes on or off.

The project’s data revealed that the strongest epigenetic signal was found in “preadipocyte” cells, the progenitor cells that go on to become fat cells.

Melina Claussnitzer. Image: Lovely Valentine“Previous studies attempted to uncover a link between FTO and the regulation of appetite or propensity to exercise controlled by the brain,” said the study’s lead and corresponding author Melina Claussnitzer, an HMS instructor in medicine and an investigator in the Division of Gerontology at Beth Israel Deaconess and Hebrew SeniorLife, a visiting professor at MIT’s Computer Science and Artificial Intelligence Laboratory (CSAIL), and member of the Broad Institute.

“But an unbiased look across more than one hundred human tissues and cell types indicated that the obesity-associated region acts primarily in adipocyte progenitor cells—not the brain,” she said.

The researchers collected fat, or adipose, tissue samples from individuals who carried the genetic obesity risk variant and compared it with tissue samples of individuals who did not carry the variant; they found increased expression of two distant genes, IRX3 and IRX5, indicating that these genes are under genetic control by the obesity risk variant.

“Despite years of investigating the FTO obesity region, no substantial expression differences were found between obesity-risk and non-risk individuals in brain or other tissue types, making it difficult to trace its mechanism of action,” said Manolis Kellis, professor at MIT’s CSAIL.

“We found a strong difference for both IRX3 and IRX5 in preadipocytes, revealing the target genes, cell type and developmental stage where the genetic variant acts, thus enabling us to begin dissecting its mechanism of action,” said Kellis.

Manipulating the pathways

Elevated expression of these genes resulted in a shift from energy-burning beige fat cells to energy-storing white fat cells. The researchers showed that they could manipulate this new pathway to reverse the signatures of obesity.

“By altering the expression of either gene in human preadipoctyes, we could alter adipocyte metabolism between energy storage and energy dissipation, providing a direct link between IRX3 and IRX5 expression and energy balance,” said Kellis.

To evaluate the effect of IRX3 inhibition on whole-body energy metabolism and body weight, the team inhibited the corresponding gene in the fat cells of mice. The animals’ metabolism increased and they lost weight, even though their physical activity and appetite were unchanged.

“The results at the organism level were dramatic,” said Claussnitzer.

“These mice were 50 percent thinner than the control mice, and they did not gain any weight on a high-fat diet. Instead they dissipated more energy, even in their sleep, suggesting a dramatic shift in their global metabolism. The circuitry underlying the FTO region functions like a master regulatory switch between energy storage and energy dissipation,” she said.

The researchers then sought to connect these differences in metabolism and gene expression to the genetic differences between lean and obese people within the FTO gene.

They predicted that the specific T-to-C single-nucleotide alteration within FTO is responsible for the obesity association by repressing an evolutionarily conserved gene regulator called ARID5B.

Loss of repression turns on IRX3 and IRX5 during early adipocyte differentiation, leading to a shift from beige adipocyte functions and thermogenesis, or energy burning, to white adipocyte lipid accumulation.

“We could narrow down a genetic region spanning 47,000 nucleotides to reveal a single-nucleotide alteration, and explain precisely how it leads to loss of repressor binding, activation of a regulatory region, gain of distal gene expression, a change in adipocyte metabolism, and ultimately, obesity at the organism level,” explained Claussnitzer.

Noncoding variants

“This can serve as a model for understanding the mechanistic basis of other non-coding variants in other diseases and traits. Noncoding variants make up more than 90 percent of top-scoring variants that have emerged from genome-wide association studies, which find association between genetic variants and disease risk, she said.”

Using the genome editing technique known as CRISPR/Cas9, the team found that switching the risk variant to the protective variant in preadipocytes turned off IRX3 and IRX5 and restored thermogenesis, while the reverse change turned on IRX3 and IRX5 and turned off thermogenesis.

“Bidirectional genome editing of the causal nucleotide variant allowed us to demonstrate that a single nucleotide is responsible for flipping this metabolic switch between obese and lean individuals,” said Claussnitzer.

“This is the first time that causality has been demonstrated for a genetic variant in a distal non-coding region, but we hope it will be the first of many such studies to come, now that genome editing is becoming broadly adopted,” she added.

This study was supported, in part, by grants from National Institutes of Health (R01HG004037, R01GM113708 and RC1HG005334).

Adapted from a Beth Israel Deaconess news release. 


Gene for Mitral Valve Prolapse

International network reveals role of gene mutations in families with inherited cardiac disorder

An international research collaboration led by Harvard Medical School investigators at Massachusetts General Hospital has identified the first gene whose mutations cause the common form of mitral valve prolapse, a heart valve disorder that affects almost 2.5 percent of the U. S. population.

In a paper published in Nature, the team reports finding mutations in a gene called DCHS1 in affected members of three families in which mitral valve prolapse is inherited.

“This work provides insights into the pathways regulating valve growth and development and implicates a previously unrecognized basis for the long-term structural integrity of the mitral valve,” said senior author Susan Slaugenhaupt, HMS professor of neurology.

Mitral valve prolapse. Image: NIHLBIShe is also scientific director of the Mass General Research Institute, an investigator at the Mass General Center for Human Genetic Research and one of the lead scientists in the collaborative group that conducted the research. 

“This finding can teach us how to prevent this inborn disease from manifesting as an illness in people who inherit mutated forms of this gene,” said Robert Levine, HMS professor of medicine at Mass General and co-senior author of the Nature paper. “Understanding how defects in this gene cause errors in early valve formation can point to ways we can prevent the progression of this condition to keep the valve and the heart healthy and help the patient avoid complications.”

One of four valves controlling the flow of blood through the heart, the mitral valve lies between the left atrium and the left ventricle, which handle oxygenated blood returning from the lungs. The valve consists of two leaflets that open to let blood pass through and close to keep it from moving backwards.

In mitral valve prolapse, the leaflets become thickened, elongated and floppy, preventing the valve from closing completely and allowing blood to leak backward in a process called regurgitation. Patients with serious mitral valve prolapse can develop shortness of breath, cardiac arrhythmia, heart failure or an infection of the heart valves. Mitral valve prolapse is the most common reason for mitral valve surgery.

While mitral valve prolapse can accompany connective tissue disorders such as Marfan syndrome, its most common form runs in families without such syndromes. A specific genetic cause of familial mitral valve prolapse had not previously been identified, and the Mass General research team’s first step was to link the occurrence of mitral valve prolapse in one large family to a genetic risk factor located on chromosome 11.

Their work depended on advanced diagnostics developed by Levine and colleagues in the Mass General Cardiac Ultrasound Laboratory, which in turn relied on their discovery of the three-dimensional shape of the mitral valve.

In the current study, detailed DNA analysis of the affected members of that family identified two rare mutations in DCHS1, a chromosome 11 gene previously studied in fruit flies. Experiments in zebrafish led by David Milan, HMS assistant professor of medicine at Mass General and co-senior author of the paper, revealed that inactivation of an analogous gene led to significant defects in the development of the heart at the site corresponding to the mitral valve. These defects could be prevented by the introduction of the normal copy of the human DCHS1 gene but not the mutated version.

Based on those findings, the Mass General team sought to collaborate with other groups studying mitral valve prolapse in order to determine whether the same mutations had a role in other families with the condition. A major grant from the Leducq Foundation enabled the formation of a network consisting of 11 centers in the U.S. and four European countries. Analysis of DNA from those centers identified two French families in which mitral valve prolapse was caused by another DCHS1 mutation.

Further experiments in cells indicated that mutations associated with mitral valve prolapse significantly reduced the expression of the DCHS1 protein, which helps organize how cells are patterned into tissues.

Co-senior author Russell Norris and his team at the Medical University of South Carolina analyzed the development of mice in which one copy of the DCHS1 gene was mutated, as it is in affected family members. Their findings revealed that the gene plays a critical role in the proper formation of the mitral valve, the first evidence for the gene’s role in cardiac development, and that the mutation led to mitral valve changes resembling the human disease.

“This discovery required the cooperation of multiple disciplines and teams—ranging from clinical cardiology and ultrasound diagnostics to classical genetics, screening of potential mutations in zebrafish and functional studies in our mouse models,” said Levine.

Milan added, “As a follow-up, this same international network has been seeking other genes that cause mitral valve prolapse across the population, which should point us to common pathways that could be targets of therapies designed to prevent progression into symptomatic disease.”

In addition to a grant from the Leducq Foundation, support for the study includes grants from the Doris Duke Medical Foundation and several grants from the National Heart, Lung, and Blood Institute.

Adapted from a Mass General news release.


Building Muscle

Lab-grown muscle fibers offer new muscular dystrophy model 

Skeletal muscle is one of the most abundant tissue types in the human body, but it has proven difficult to produce in large quantities in the lab.

Unlike other cell types, such as heart cells, neurons and cells found in the gut, previous attempts to efficiently and accurately derive muscle cells from precursor cells or from culture have not been fruitful.

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In a study published Aug. 3 in Nature Biotechnology, a research team led by investigators from Harvard Medical School, Brigham and Women’s Hospital and the Harvard Stem Cell Institute report that by identifying and mimicking important developmental cues, they have been able to drive cells to grow into muscle fibers, producing millimeter-long muscle fibers capable of contracting in a dish and multiplying in large numbers.

This new method of producing muscle cells could offer a better model for studying muscle diseases, such as muscular dystrophy, and for testing potential treatment options.

Previous studies have used genetic modification to create small numbers of muscle cells in the lab, but the research team wanted a technique that would allow them to grow large numbers of muscle cells efficiently for use in clinical applications.

“We took the hard route,” said corresponding author Olivier Pourquié, the Frank Burr Mallory Professor of Pathology at Brigham and Women’s and professor of genetics at HMS. “We wanted to recapitulate all of the early stages of muscle cell development that happen in the body and recreate that in a dish in the lab.”

“We analyzed each stage of early development and generated cell lines that glowed green when they reached each stage,” he said. “Going step by step, we managed to mimic each stage of development and coax cells toward muscle cell fate.”

The team found that a combination of secreted factors that are important at early embryonic stages are also essential for stimulating differentiation—the specialization of stem cells into particular cell types—in the lab. Using just the right recipe for differentiation, the team was able to produce long, mature fibers in a dish, derived from mouse or human pluripotent stem cells.

They also cultured stem cells from a mouse model of Duchenne muscular dystrophy, observing the striking branched pattern that dystrophin-deficient muscle fibers show in the body.

The research team was also able to produce more-immature cells known as satellite cells which, when grafted into the mouse model of Duchenne muscular dystrophy, produced muscle fibers. Further studies will be needed to determine if the new strategy could be optimized to develop cell therapies for treating degenerative diseases in humans.

In addition to developing a better model for Duchenne muscular dystrophy, the new protocol may be useful for studying other muscle diseases such as sarcopenia (degenerative muscle loss), cachexia (wasting away of muscle associated with severe illness) and other muscular dystrophies.

“This has been the missing piece. The ability to produce muscle cells in the lab could give us the ability to test out new treatments and tackle a spectrum of muscle diseases,” said Pourquié.

This work was supported by the European Research Council, the Stowers Institute for Medical Research, the Howard Hughes Medical Institute, the FP7 EU grant Plurimes (agreement no. 602423) and a strategic grant from the French Muscular Dystrophy Association (AFM).

Adapted from a Brigham and Women’s news release.


Delivering Hope in Ovarian Cancer

Gene therapy blocked chemoresistant tumor growth in mice

Using gene therapy to deliver a protein that suppresses the development of female reproductive organs may improve the survival of patients with ovarian cancer that has recurred after chemotherapy. Recurrence happens 70 percent of the time and is invariably fatal. 

In their report published in PNAS Early Edition, Harvard Medical School researchers at Massachusetts General Hospital describe how a single injection of a modified version of Mullerian Inhibiting Substance, a protein critical to sexual development, suppressed the growth of chemotherapy-resistant ovarian tumors in a mouse model. 

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While not all the tested tumors, which were grown from cells grafted from patient tumors, were sensitive to this treatment, the investigators also outlined a noninvasive way to screen cancer cells in vitro for treatment responsiveness.

“Our findings are important because there are currently no therapeutic options for recurrent, chemoresistant ovarian cancer,” said David Pépin, HMS instructor in surgery at Mass General and lead author of the report. “This is also a proof of concept that gene therapies with the AAV9 vector can be used to deliver biologics for the treatment of ovarian cancer and represents the first time this approach has been tested in this type of ovarian cancer model.” 

Potential to treat cancer

During embryonic development, Mullerian Inhibiting Substance is secreted by tissues in male embryos to prevent maturation of the Mullerian duct, which otherwise would give rise to female reproductive organs. The potential of MIS to treat ovarian cancer and other reproductive tumors has been studied for many years by Patricia Donahoe,  Marshall K. Bartlett Distinguished Professor of Surgery at HMS, director of Mass General’s Pediatric Surgical Research Laboratories and senior author of the current study. 

Previous investigations by Donahoe’s team showed that MIS suppresses ovarian cancer growth, both in animals and in human cell lines, by targeting the cancer stem cells that survive chemotherapy. But previous methods of producing MIS were unable to generate sufficient quantities of high-quality protein for pre-clinical testing.

The current study employed a modified form of the MIS gene, developed by Pépin to generate proteins of greater purity and effectiveness, combined with the accepted viral vector AAV9 for delivery into the peritoneal cavity, a common site for the recurrence of ovarian cancer. The modified MIS/AAV9 construct was tested against tumor cells taken from ascites fluid that had accumulated within the abdomens of several patients with recurrent ovarian cancer. 

Testing effectiveness

Initial experiments confirmed that these cells expressed the MIS receptor protein, that they carried markers indicating their identity as cancer stem cells, and that their growth was inhibited in vitro by MIS. A single injection of the MIS/AAV9 construct into the peritoneal cavity of mice resulted in elevated expression of MIS by multiple tissues throughout the abdominal cavity and in adjacent muscles.

The effectiveness of the MIS/AAV9 construct was tested in mice into which ovarian cancer cells were implanted. Those efforts revealed that treatment with MIS/AAV9 three weeks before tumor implantation significantly inhibited tumor growth. 

In a more clinically relevant experiment, the therapy was applied to mice in which tumors already had been induced by implantation of cancer cells from five different patients. The therapy resulted in significant inhibition of further growth of tumors generated from the cells of three of the five patients. 

Analysis of tumor samples from more than 200 patients revealed that 88 percent expressed some level of the MIS receptor, with 65 percent expressing moderate or high protein expression.

Need for biomarkers

“Since the response to MIS gene therapy is not the same for all patients, it will be important to first screen each patient’s tumors to ensure they will respond” said Pépin. “While we have not yet identified biomarkers of treatment response—something we are currently searching for—we have described a way to rapidly grow tumor cells from ascites to be evaluated for drug sensitivity. If further study confirms the susceptibility of chemoresistant tumors to this MIS gene therapy, the ability to inhibit tumor recurrence could significantly extend patient survival.”

The MIS/AAV9 construct was prepared by Guangping Gao, director of the Gene Therapy Center at the University of Massachusetts Medical School.  The single, long-acting injection makes the use of this effective but complex protein both clinically feasible and patient friendly, Donahoe said.

“All of the implanted tumor cells were from patients who failed all previous therapies, so a 60 percent response rate is quite significant for a single agent,” said Donahoe. “Our results provide proof of concept and predict a translation into patient care that was not previously possible.”

Support for the study includes grants from the Ovarian Cancer Research Fund, the Sudna Gar Foundation, Department of Defense Award OC110078 and National Institutes of Health grant R01-CA17393.

Adapted from a Mass General news release.


American History 201

Genetic studies link indigenous peoples in the Amazon and Australasia

Native Americans living in the Amazon bear an unexpected genetic connection to indigenous people in Australasia, suggesting a previously unknown wave of migration to the Americas thousands of years ago, a new study has found.

“It’s incredibly surprising,” said David Reich, Harvard Medical School professor of genetics and senior author of the study. “There’s a strong working model in archaeology and genetics, of which I have been a proponent, that most Native Americans today extend from a single pulse of expansion south of the ice sheets—and that’s wrong. We missed something very important in the original data.”

Previous research had shown that Native Americans from the Arctic to the southern tip of South America can trace their ancestry to a single “founding population” called the First Americans, who came across the Bering land bridge about 15,000 years ago. In 2012, Reich and colleagues enriched this history by showing that certain indigenous groups in northern Canada inherited DNA from at least two subsequent waves of migration.

First author Pontus Skoglund explains his team’s new hypothesis about the ancient migrations that gave rise to today’s Native Americans. Video: Stephanie Dutchen

The new study, published July 21 in Nature, indicates that there’s more to the story.

Pontus Skoglund, first author of the paper and a postdoctoral researcher in the Reich lab, was studying genetic data gathered as part of the 2012 study when he noticed a strange similarity between one or two Native American groups in Brazil and indigenous groups in Australia, New Guinea and the Andaman Islands.

“That was an unexpected and somewhat confusing result,” said Reich, who is also an associate member of the Broad Institute of Harvard and MIT and a Howard Hughes Medical Investigator. “We spent a really long time trying to make this result go away and it just got stronger.”

Skoglund and colleagues from HMS, the Broad and several universities in Brazil analyzed publicly available genetic information from 21 Native American populations from Central and South America. They also collected and analyzed DNA from nine additional populations in Brazil to make sure the link they saw hadn’t been an artifact of how the first set of genomes had been collected. The team then compared those genomes to the genomes of people from about 200 non-American populations.

The link persisted. The Tupí-speaking Suruí and Karitiana and the Ge-speaking Xavante of the Amazon had a genetic ancestor more closely related to indigenous Australasians than to any other present-day population. This ancestor doesn’t appear to have left measurable traces in other Native American groups in South, Central or North America.

The genetic markers from this ancestor don’t match any population known to have contributed ancestry to Native Americans, and the geographic pattern can’t be explained by post-Columbian European, African or Polynesian mixture with Native Americans, the authors said. They believe the ancestry is much older—perhaps as old as the First Americans.

In the ensuing millennia, the ancestral group has disappeared.

“We’ve done a lot of sampling in East Asia and nobody looks like this,” said Skoglund. “It’s an unknown group that doesn’t exist anymore.”

The team named the mysterious ancestor Population Y, after the Tupí word for ancestor, “Ypykuéra.”

Reich, Skoglund and colleagues propose that Population Y and First Americans came down from the ice sheets to become the two founding populations of the Americas.

“We don’t know the order, the time separation or the geographical patterns,” said Skoglund.

Researchers do know that the DNA of First Americans looked similar to that of Native Americans today. Population Y is more of a mystery.

“About 2 percent of the ancestry of Amazonians today comes from this Australasian lineage that’s not present in the same way elsewhere in the Americas,” said Reich.

However, that doesn’t establish how much of their ancestry comes from Population Y. If Population Y were 100 percent Australasian, that would indeed mean they contributed 2 percent of the DNA of today’s Amazonians. But if Population Y mixed with other groups such as the First Americans before they reached the Americas, the amount of DNA they contributed to today’s Amazonians could be much higher—up to 85 percent.

To answer that question, researchers would need to sample DNA from the remains of a person who belonged to Population Y. Such DNA hasn’t been obtained yet. One place to look might be in the skeletons of early Native Americans whose skulls some researchers say have Australasian features. The majority of these skeletons were found in Brazil.

Reich and Skoglund think that some of the most interesting open questions about Native American population history are about the relationships among groups after the initial migrations.

“We have a broad view of the deep origins of Native American ancestry, but within that diversity we know very little about the history of how those populations relate to each other,” said Reich.

This work was supported by the National Science Foundation (HOMINID grant BCS-1032255), the National Institutes of Health (GM100233), the Simons Foundation (grant 280376), the Howard Hughes Medical Institute, the Conselho Nacional de Desenvolvimento Científico e Tecnológico and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil), the Wenner-Gren Foundation and the Swedish Research Council (VR grant 2014-453). 


Evolving CRISPR

Engineered variants double the target range and improve the precision of gene-cutting enzyme

A team of Harvard Medical School researchers at Massachusetts General Hospital has found a way to expand the use and precision of powerful gene-editing tools called CRISPR-Cas9 RNA-guided nucleases.

In their report in Nature, the investigators describe evolved versions of the DNA-cutting Cas9 enzyme that can recognize a different range of nucleic acid sequences than is now possible with the naturally occurring form of Cas9 scientists have been using.

“In our paper we show that sites in human and zebrafish genes that could not previously be modified by wild-type Cas9 can now be targeted with the new variants we have engineered,” said Benjamin Kleinstiver, HMS research fellow in pathology at Mass General and lead author of the Nature paper. “This will allow researchers to target an expanded range of sites in a variety of genomes, which will be useful for applications requiring highly precise targeting of DNA sequences.”

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CRISPR-Cas9 nucleases consist of the Cas9 bacterial enzyme and a short, 20-nucleotide RNA molecule that matches the target DNA sequence. In addition to the RNA/DNA match, the Cas9 enzyme needs to recognize a specific nucleotide sequence called a protospacer adjacent motif (PAM) adjacent to the target DNA.

The most commonly used form of Cas9, derived from the bacteria Streptococcus pyogenes and known as SpCas9, recognizes PAM sequences in which any nucleotide is followed by two guanine DNA bases. This limits the DNA sequences that can be targeted using SpCas9 to only those that include two sequential guanines.

To get around this limitation, the team set up an engineering system that allowed them to rapidly evolve the ability of SpCas9 to recognize different PAM sequences. From a collection of randomly mutated SpCas9 variants, they identified combinations of mutations that enabled SpCas9 to recognize new PAM sequences.

These evolved variants essentially double the range of sites that can now be targeted for gene editing using SpCas9. They also identified an SpCas9 variant that was less likely to induce the off-target gene mutations sometimes produced by CRISPR-Cas9 nucleases, a problem originally described in a 2013 study led by J. Keith Joung, HMS professor of pathology at Mass General and senior author of the current study.

“This additional evolved variant with increased specificity should be immediately useful to all researchers who currently use wild-type SpCas9 and should reduce the frequencies of unwanted off-target mutations,” Joung said. “Perhaps more important, our findings provide the first demonstration that the activities of SpCas9 can be altered by directed protein evolution.”

The scientists showed in their paper that the forms of Cas9 found in two other bacteria—Staphylococcus aureus and Streptococcus thermophiles—can also function in their bacterial evolution system, suggesting that their functions can be modified as well, Joung said.

“This work just scratches the surface of the range of PAMs that can be targeted by Cas9,” Joung said. “We believe that other useful properties of the enzyme may be modified by a similar approach, allowing potential customization of many important features.”

The study was supported by National Institutes of Health Director’s Pioneer Award DP1 GM105378, NIH grants R01 GM107427 and R01 GM088040, a Jim and Ann Orr Research Scholar Award, and a National Sciences and Engineering Research Council of Canada Postdoctoral Fellowship. The MGH has filed a patent application on the use of the SpCas9 variants described in this paper.

Adapted from a Mass General news release.


FISHing for Insight

Improved imaging illuminates chromosomes in detail

HMS professor of genetics Ting Wu explains why she’s excited about new chromosomal imaging techniques. Video: Stephanie Dutchen

Geneticists just got a new pair of glasses.

By improving an imaging technology called FISH, they’ve made it possible to view genetic material in more detail than ever before.

One modification achieves “super resolution” imaging. The other can distinguish maternal from paternal chromosomes.

What the researchers are starting to see promises to help them better understand how DNA gets packaged into chromosomes, how that structure relates to health and disease, what the biological significance may be when genetic material is inherited from one parent versus the other—and more.

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“Geneticists have looked at chromosomes for over a century, but they’ve longed for higher resolution and an easier and more affordable strategy for looking at any part of any genome, not just the most accessible regions,” said Ting Wu, professor of genetics at Harvard Medical School and senior author of the study, published in Nature Communications. “With this new technology, we’ve enabled significant steps forward in all those regards.”

“Scientists have a lot of models where we draw in cartoon form what we think is happening,” said the study’s first author, Brian Beliveau, who conducted the work as a graduate student in the Wu lab. “When a region of the genome is silenced, we draw it as compacted. When a gene is expressed, we draw it as more open or active. But we don’t actually know yet what any of these look like.”

“Now we have a tool to start looking at this systematically, which is very exciting,” said Beliveau, currently a research fellow in the lab of HMS associate professor of systems biology Peng Yin at the Wyss Institute for Biologically Inspired Engineering.

Brian Beliveau describes the advances Oligopaints have made possible. Video: Stephanie Dutchen

Teach a person to FISH…

FISH, short for fluorescence in situ hybridization, is a decades-old visualization technique that locates and illuminates specific genes in the nuclei of cells.

Scientists use FISH to find out things like how many chromosomes a person has or where a particular gene is located on a chromosome.

But conventional FISH has its limits, and scientists want to look closer.

For instance, it hasn’t been able to show the details of how chromosomes are folded, whether there are different ways of folding or how two genome segments interact—all of which would help elucidate how our bodies work.

Researchers have had some success getting high-resolution FISH images of small segments of the genome. But the cost to look at such questions genome-wide has been “beyond anybody’s purse,” said Wu. “The flexibility to target a specific region on the genome has been very difficult also.”

In 2012, Beliveau devised an enhancement to FISH called Oligopaint that made this possible at low cost.

Traditional FISH works by attaching a fluorescent tag to a short, single strand of DNA called a probe, which has a sequence complementary to the segment of DNA a researcher wants to study. When released into a cell, the probe binds to the desired sequence in the nucleus and lights it up so it can be seen under a microscope.

A limiting factor to scaling up FISH has been that “it’s a ton of work to try to find those sequences in nature to make the necessary DNA probes and isolate them in a way that would be compatible with the tool,” said Beliveau.

He solved the problem by developing computer software that lets scientists design the probes they need and then build them with synthetic components—hundreds or thousands at a time.

Super resolution

In the new paper, Beliveau taught Oligopaint two new tricks.

First, he paired it up with two other technologies—STORM from the lab of Xiaowei Zhuang at Harvard University, and DNA-PAINT from Peng Yin’s lab—to zoom in on chromosomes at “super resolution.”

Conventional (left) and STORM (right) images depict the same field of view at the same magnification within a cell. STORM reveals two loop-like protrusions in the cell’s genetic material. Image: Alistair Boettiger, Zhuang lab

The DNA double helix is about 2 nanometers wide. When it gets wound around a bundle called a nucleosome, one of the simplest building blocks of chromosomes, it grows to about 10 nanometers. Since conventional microscopy can resolve images to only about 200 to 300 nanometers, it’s been impossible to see such infinitesimal structures.

The new Oligopaint combinations can bring chromosomes into focus down to about 20 nanometers. That’s possible because each DNA probe is able to bind to a second probe; the resulting fluorescent output blinks, enabling researchers to distinguish the fluorescent tags one at a time and achieve a finer resolution.

“This is an unprecedented level of detail,” said Wu.

“Right now when you do FISH on structures below a certain size, you get a spot,” said Beliveau. “With these technologies, we’re seeing interesting shapes and structures, loops and protrusions, start to drop out of these things.”

Wu, Beliveau and their colleagues have already begun to find “intriguing organizational themes” in the chromatin of mammalian and fruit fly cells. But they consider the current paper mostly a proof of concept.

“The goal is to communicate these techniques to the research community as quickly as possible so everyone can start making discoveries,” said Beliveau.

They hope researchers generate “a huge bolus of images” that will reveal more about what our genetic structure looks like, Wu added.

“Of course, naturally, if we get down to 20 nanometers then people will want 10 nanometers, and then 5,” she said wryly.

Distinguishing mom from dad

We normally inherit our chromosomes pairs: one from our mother and one from our father. The second trick Beliveau taught Oligopaint was to tell them apart.

“This was not thought possible but, to our surprise, our technology managed to break this barrier too,” said Wu.

Beliveau modified his Oligopaint probes so they could detect the presence or absence of the many single nucleotide variants, or SNPs, that distinguish maternal from paternal chromosomes.

As long as researchers know which SNPs to look for and have hundreds or thousands of them to target—just a few isn’t enough—Oligopaint can now light up a chromosome to identify “mom” or “dad.”

Wu, for one, is excited to use this new ability to study things like how one of two X chromosomes gets inactivated in female mammals. It’s also become clear to scientists that maternal and paternal genes don’t behave the same way, and that this might have effects on human health and disease. Wu is particularly interested in Down syndrome, in which children inherit three instead of two copies of chromosome 21.

“I’d love to look at a cell and ask, are two copies from the mother? From the father? How do they behave relative to each other?” she said.

This study was supported by the National Institutes of Health (R01GM61936, R01GM090278, 1R01EB018659, 5DP1GM106412, F32CA157188, 1DP2OD007292, 5R21HD072481, 1DP2OD004641), National Science Foundation (CCF1054898, CCF1162459), Office of Naval Research (N000141110914, N000141010827, N000141310593), Harvard Medical School, Wyss Institute for Biologically Inspired Engineering, Centre National de la Recherche Scientifique, Howard Hughes Medical Institute, Damon Runyon Cancer Research Foundation, Fulbright Visiting Scholar Program and Alexander von Humboldt-Foundation.