A team led by Harvard Medical School and Massachusetts General Hospital investigators has found that the controlled induction of the hypoxia response, the body’s reaction to a reduced level of oxygen in the bloodstream, may relieve the symptoms of one of the most challenging groups of genetic disorders—mitochondrial diseases.
Their report describing experiments in cellular and animal models of mitochondrial disease has been published online in the journal Science.
“We reasoned that reducing the level of oxygen the mice breathed and delivered to their tissues might serve as nature’s perfect solution for mitochondrial disease,” -- lead author Isha Jain.
“We currently lack effective means of treating mitochondrial diseases, of which there are more than 150 different genetic forms, impacting virtually any organ,” said Vamsi Mootha, HMS professor of systems biology, a researcher in the Mass General Department of Molecular Biology and senior author of the Science report.
“We found that activation of the hypoxia response—either genetically or pharmacologically in cells, or by placing mice in a low-oxygen environment—alleviated mitochondrial pathology,” he said.
Many mitochondrial diseases appear in infancy or early childhood, but in others symptoms may not appear until adulthood and are often triggered by external stressors such as infection.
Although the mitochondria in many organs are defective in these disorders, disease pathology is manifest only in certain tissues. These observations led the researchers to hypothesize that there might be inborn mechanisms for coping with mitochondrial dysfunction.
They created a cellular model in which respiratory chain activity was inhibited by application of a toxin and used the model to test whether disruption of any of about 18,000 different genes alleviated the cellular effects of mitochondrial dysfunction.
The top-ranking gene in their analysis was for Von Hippel Lindau factor (VHL), which ordinarily suppresses the cellular response to hypoxia, implying that the body’s natural hypoxia response might protect against mitochondrial injury.
The researchers then showed that both cells and embryonic zebrafish that lacked VHL exhibited greater survival in the face of respiratory chain inhibition.
Treatment with a chemical that increases the expression of genes involved in the hypoxia response also reduced death from respiratory chain inhibition.
The researchers next graduated to rodent studies, using an accurate genetic model of a specific mitochondrial disease. Mootha’s team partnered with Warren M. Zapol, the HMS Reginald Jenney Professor of Anaesthesia at Mass General.
The collaborative team focused on the Ndufs4-knockout mouse, an established model of Leigh syndrome, a neurodegenerative condition that is the most common pediatric manifestation of mitochondrial disease.
They first showed that this model could survive brief periods of hypoxia with a relatively normal metabolic response and then tested the effects of keeping the animals in an atmosphere containing 11 percent oxygen—similar to high-altitude environments like the mountains of Nepal and Peru—for extended periods of time.
A control group of Ndufs4-knockout mice was placed in ambient air containing about 21 percent oxygen.
“We reasoned that reducing the level of oxygen the mice breathed and delivered to their tissues might serve as nature’s perfect solution for mitochondrial disease,” said lead author Isha Jain, a graduate student in the Mass General Department of Molecular Biology.
“After all, humans have evolved elaborate responses to cope with energy metabolism at high altitudes and low oxygen levels.”
The researchers were pleasantly surprised to discover that breathing 11 percent oxygen—approximately half what is normally supplied at sea level—dramatically reduced the development of typical disease symptoms, such as restricted growth and neurologic and movement abnormalities, and significantly extended survival in the Ndufs4-knockout mice.
While knockout animals breathing air with 21 percent oxygen either died or had symptoms severe enough to require humane euthanasia by 60 days of age, all of those in the low-oxygen environment survived to at least 150 days, and some have survived up to 250 days.
In contrast, although normal animals easily tolerated a 55 percent oxygen environment, Ndufs4-knockout mice placed in the same high-oxygen environment died within 2 to 11 days.
“Many species of animals live at high altitudes around the world and adapt to low oxygen levels by activating specialized molecular systems,” said Zapol, who is a pioneer in the therapeutic use of gas mixtures.
“While it might be expected that mice with a mitochondrial defect would have even more trouble generating ATP for cellular energy in a low-oxygen environment, we were surprised that they seemed healthy and didn’t show signs of oxygen starvation," he said. "It’s remarkable that, with about half the normal level of oxygen circulating in their blood, they appeared to resist the brain pathology associated with this mitochondrial defect for several months.”
Both Zapol and Mootha stress that much more research needs to be done before a hypoxia-based strategy can be tested in patients with mitochondrial disease.
“All our work to date has been limited to cells and mice, which are not humans,” said Mootha.
“Breathing hypoxic air can be dangerous and could reduce oxygen delivery to major organs as well as produce acute and chronic toxicities. Therefore, it’s crucial to perform additional animal studies to determine optimal treatment regimens and long-term safety. Moreover, there are many types of mitochondrial disease, and we have only tested one. Testing additional mouse models will help us determine whether and when it will be feasible to contemplate human trials,”Mootha said.
This work was supported in part by the Howard Hughes Medical Institute and a grant from the Marriott Mitochondrial Disorders Research Fund. Mass General has filed a patent application for the work described in this study.
Adapted from a Mass General news release.
Obesity and its associated health risks are a major health care concern in the U.S. More than 78 million American adults are considered obese, with a growing number of American children affected as well, according to the National Heart, Lung, and Blood Institute.
In 2013, NHLBI statistics indicated that about 17 percent of U.S. children and adolescents between the ages of 2 and 19 were obese—one in six. Obesity can lead to increased rates of heart disease, type 2 diabetes, high blood pressure and certain cancers.
Sir Stephen O’Rahilly, professor of clinical biochemistry and medicine at the University of Cambridge and an authority on human obesity and insulin resistance, will be a guest lecturer at the 2015 Dunham Lecture Series at Harvard Medical School on April 6 and 7.
O’Rahilly is director of the University of Cambridge Metabolic Research Laboratories, co-director of the Institute of Metabolic Science and director of the MRC Metabolic Diseases unit.
A member of the Academy of Medical Sciences and the Royal Society, O’Rahilly is one of the United Kingdom’s most renowned clinical researchers. He is widely known for combining research into the causes of obesity and insulin resistance with clinical practice.
“I have a long-standing interest in the aetiology and pathophysiology of human metabolic and endocrine disease and how such information might be used to improve diagnosis, prognostication, therapy and prevention,” O’Rahilly says on his Cambridge lab website.
According to his nomination to the Royal Society in 2003, O’Rahilly’s “work first established that mutations in a single gene could result in severe human obesity and that these defects largely acted through disruption of central satiety mechanisms.” He was knighted in 2013 for his service to medical research.
O’Rahilly’s 60-minute lecture on Monday, April 6, is entitled “Human Obesity as a Neurobehavioral Disorder: Lessons from Human Genetics.” The lecture will take place in the Joseph B. Martin Conference Center from 4 to 5 p.m. with a reception to follow.
The hour-long Tuesday, April 7, lecture is entitled “Mechanisms of Human Insulin Resistance,” and it will take place from noon to 1 p.m. in the Martin Conference Center at 77 Avenue Louis Pasteur.
Both lectures are free and open to students, faculty and staff of HMS and Harvard University, as well as other interested professionals. Seats are available on a first-come, first-served basis.
Those planning to attend may RSVP to Dunham@hms.harvard.edu by April 1, indicating which lectures they will attend.
The prestigious Dunham Lecture Series was inaugurated at HMS in 1923, established by Mary Dows Dunham in honor of her late husband, Edward Kellogg Dunham (Harvard 1886), to strengthen the bonds of fellowship and understanding among students, investigators and faculty within the medical and basic sciences, for the purpose of advancing medical science in the broadest sense.
Twenty-eight of the 78 biomedical researchers who have been honored with a Dunham lectureship have been Nobel laureates.
Following a multiyear process of self-reflection and review, Jeffrey S. Flier, Dean of Harvard Medical School, has announced the establishment of a new Department of Biomedical Informatics (DBMI). It will officially become a Quad-based department on July 1, 2015.
Isaac “Zak” Kohane, co-director of the HMS Center for Biomedical Informatics, director of the Countway Library of Medicine and the HMS Lawrence J. Henderson Professor of Pediatrics at Boston Children’s Hospital, will be the department’s inaugural chair.
“The field of biomedical informatics has its roots in a half century of academic development, in which Harvard has played a prominent role since its earliest days,” Flier said in a letter to the community.
“The field represents two converging communities: one involving health care-related data, and the other addressing the study of health and disease at a molecular and naturally occurring systems level, " he said, adding, "These two communities are jointly focused on new methods for the capture, representation, storage and analysis of big biomedical data and knowledge. Successful implementation of precise and individualized medical care will require the development of a new generation of informatics tools to guide clinicians in applying this rapidly growing base of biomedical knowledge.”
The new department evolved out of the HMS Center for Biomedical Informatics (CBMI), which was founded in 2005. Led by Kohane and Alexa McCray, HMS associate professor of medicine at Beth Israel Deaconess Medical Center, CBMI has developed an international reputation.
In 2014, the center brought in an array of federal grants that have established large collaborations with researchers on the Quad, at affiliated hospitals and at the Harvard University Faculty of Arts and Sciences.
“Based on the increasing impact of the field and the tremendous success of CBMI, we have concluded that biomedical informatics is a field now ready for full academic recognition as a new appointing department at HMS,” Flier said.
The department will launch with five core faculty members now associated with CBMI, with recruits added over the upcoming years to achieve representation from across the field.
Kohane will also chair an executive committee, with membership drawn from leaders in biomedical informatics at HMS-affiliated institutions. The committee will eventually review and recommend primary and secondary appointments to the department across the faculty.
Mastering the surge in biomedical data streams goes beyond storage and computation. It includes developing and applying new methods for both research and clinical care.
Central to the department’s mission will be creating a way to address methodological, engineering and educational challenges. CBMI already has a strong history of education through its involvement in master’s- and doctoral-level programs. The new department will continue to expand this commitment to education.
“Biomedical informatics has the potential to transform biomedical research and human health in ways that we could not have imagined only a few years ago,” said Flier.
“I am excited to launch this new department, and I’m grateful to Provost Alan Garber, HMS preclinical department chairs, affiliated hospital leaders, and the external advisory group who worked with our HMS team, to evaluate how such a department could most effectively advance the ambitious research and clinical missions of our School,” Flier said.
Members of the Harvard Medical School Biomedical Informatics Advisory Group are Edward Frymoyer, president of Frymoyer Holdings, Inc.; Jeff Hammerbacher, founder and chief scientist of Cloudera; Gilbert Omenn, director of the center for computational medicine and at the University of Michigan; Jim Reese, former chief operations engineer and “head neurosurgeon” at Google, Inc.; and Halle Tecco, cofounder and managing director of Rock Health.
The music was so loud Eileen Sun could feel the drums thrumming in her chest. With a dozen fellow students, she was witnessing a traditional healing ceremony in rural South Africa, part of fieldwork for TransMed, a Harvard Medical School summer course on translational medicine offered through the HMS Office of Global Programs.
The healers danced, calling on spirits to help their patients. They presented scarves in vibrant colors to their guests before sharing a feast with them—one for which a cow had been sacrificed earlier in the week.
Sun saw firsthand how TransMed’s hosts at Edendale Hospital in KwaZulu-Natal were bridging the gap between Western medicine and traditional healing. In the countryside, the healers are the first choice for many people when they fall ill.
In a region where 37.4 percent of the population lives with HIV infection, these healers have been enlisted by a nonprofit program at the hospital, iTEACH (Integration of TB in Education and Care for HIV/AIDS), ), run by Krista Dong, HMS lecturer on medicine at Massachusetts General Hospital, to refer people to physicians for testing and, if the results are positive, to encourage them to take antiretroviral medications.
It took respectful relationship building to bridge the gulf between traditional and modern medicine. Sun saw a similar spirit of collaboration in FRESH (Females Rising through Empowerment, Support, and Health). The program enrolls young women when they are at highest risk for HIV infection in a study that screens their blood samples for potential infection and in exchange offers them education and employment skills training. The project is a collaboration with the Ragon Institute of MGH, MIT and Harvard.
“Behind each sample, there’s a story,” said Sun, a doctoral student studying RNA viruses at Harvard’s Program in Virology and in the Leder Human Biology and Translational Medicine Program. “The trip opened up a whole new dimension to how I’ve viewed scientific research. We saw firsthand how weaving science with a social mission—women’s empowerment—can be life changing for both researchers and research participants. The trip was inspirational.”
The students’ trip to South Africa followed two weeks of boot camp-style classes at HMS.
The mission of the course: to teach students how to assess unmet medical needs, follow the discovery process that uncovers the causes of disease, and examine how disease is detected, diagnosed and treated.
On the HMS Quad, students learned about the gap between basic discoveries and approved therapies. “The valley of death” is a term used in drug development for the abyss where promising drug leads can languish, never reaching the people they were intended to help.
The TransMed course brought in experts from the biotech and pharmaceutical industries who had traversed that chasm. They shared their stories in the inaugural session of a course that its director, Jagesh Shah, hopes will be repeated next summer.
It all started with identifying unmet medical needs.
“The basic scientists, the clinicians, the industry people and the regulatory people—every single one of these groups has a very different view of what an unmet medical need is,” said Shah, HMS associate professor of systems biology. “That was a design principle of the course.”
Compressed into three weeks, 24 students learned how disease mechanisms are discovered and how the drug discovery pipeline is primed to treat them. Students came not only from Harvard but also from China, South Africa and Zimbabwe.
On the Quad, the students rotated through four perspectives—basic scientist, clinician, industry professional or regulatory scientist—as they learned about three drugs (statins, Vioxx and Gleevec) and two diseases (Alzheimer’s and polycystic kidney disease).
The course also addressed how drug resistance, a well-known problem in infectious disease as well as cancer, plays out differently depending on where the patient lives.
Don Coen, HMS professor of biological chemistry and molecular pharmacology, led the discussion during a session in which HMS faculty presented cases on drug resistance, including one presented by Jonathan Li, HMS assistant professor of medicine at Brigham and Women’s Hospital, on drug-resistant mutations in the virus that causes AIDS. Certain of these mutant viruses are resistant to some older HIV drugs, but have decreased ability to replicate in the body.
The students from Africa pointed out that those older drugs are still used there because newer ones are not available for doctors to prescribe.
“Even though the viruses will evolve to be resistant to these drugs, the patient will be less ill than if you didn’t do anything, largely because of the decreased replication. In this country you would use newer combinations of drugs,” Coen said. “These drugs are clearly being used in the developing world setting. So in this course, we got the different perspectives of people on the ground in Africa, which was really enlightening.”
Felix Manyeruke, a physician from Zimbabwe who plans to specialize in respiratory medicine, sought out the course to explore translational medicine and the role of clinicians doing basic science and clinical research in developing countries.
“I learnt how we as clinicians should be involved in identifying clinical problems and how we can interact with researchers in basic sciences and engineering to be able to come up with a solution which can be used to benefit the patient,” he said in an email interview. “Patient advocacy was one new area which I was exposed to and I got to appreciate its value in driving drug development and drug regulation.”
Shah, the course director, said all the students are hungry to learn what they can do to advance drug discovery and diagnostic tool development to help patients wherever they live.
“We think about making more drugs that work, but actually if we didn’t make as many drugs that didn’t work, we would also save a lot of money,” said Shah, who favors borrowing “failure analysis,” a method widely used in engineering disciplines to learn from mistakes.
The TransMed course itself was an experiment.
“Strictly speaking, in three weeks, no one’s an expert,” Shah said. “But at the end, the students told us in their assessments that they had a better sense that the path for each drug is slightly different, but each study of how it works is an important scientific problem unto itself.”
Sun, who is in her sixth year as a doctoral student in virology, said TransMed is pushing her closer to translational research.
“Having participated in the TransMed class, I’m motivated more than ever to pursue research that will change people’s lives.”
The HMS TransMed program was supported by the Office of Global Programs and the Paul Dudley White Fund. The TransMed Global Component was facilitated by the Ragon Institute of MGH, MIT and Harvard and the HIV Pathogenesis Programme at Nelson R. Mandela School of Medicine at the University of Kwa-Zulu Natal (UKZN) in Durban, South Africa Co-Directors of Global Component are Filippos Porichis, instructor in medicine at Mass General, (Boston) and Victoria Kasprowicz, instructor in medicine at Mass General (Durban).
A therapy that liberates the immune system to attack cancer cells drove Hodgkin lymphoma (HL) into complete or partial remission in fully 87 percent of patients with resistant forms of the disease who participated in an early-phase clinical trial, Harvard Medical School investigators at Dana-Farber Cancer Institute and partnering institutions report in a study published in the New England Journal of Medicine and also presented at the annual meeting of the American Society of Hematology (ASH) in San Francisco.
The results provide some of the most dramatic evidence to date of the potential of therapies that increase the ability of the immune system to kill cancer cells. While clinical trials of such immunotherapies in other cancers have shown them to be highly effective in a subgroup of patients, the new study stands out because nearly all patients benefited from the treatment.
The success of the agent, nivolumab, in this study has prompted the U.S. Food and Drug Administration to designate it a “breakthrough therapy” for treating relapsed HL, and a large, multinational Phase 2 trial is now under way.
“What makes these results especially encouraging is that they were achieved in patients who had exhausted other treatment options,” said the study’s co-senior author Margaret Shipp, HMS professor of medicine and chief of Division of Hematologic Neoplasia at Dana-Farber. “We’re also excited by the duration of responses to the drug: The majority of patients who had a response are still doing well more than a year after their treatment.”
The study involved 23 patients with relapsed or treatment-resistant HL, a cancer of white blood cells called lymphocytes. Although relatively uncommon—with fewer than 10,000 new cases each year in the U.S.—it is one of the most frequent cancers in children and young adults. While the disease can often be treated successfully with current therapies, up to a quarter of all patients eventually have a relapse.
In the current study, almost 80 percent of the patients had undergone a previous stem cell transplant. More than a third had received at least six prior lines of therapy without lasting success.
The patients received biweekly infusions of nivolumab, which is an antibody that blocks a protein called PD-1 on the surface of immune system T cells. T cells are key actors in the body’s defenses, identifying foreign or diseased cells and leading an assault on them. But when PD-1 binds to proteins called PD-L1 and PD-L2 on the surface of certain cancer cells, the T cells essentially become paralyzed: The immune attack on cancer is called off. By blocking PD-1, nivolumab allows the attack to proceed.
“This is a treatment that, rather than targeting cancer cells themselves, targets the immune response, reactivating the T cells in the neighborhood of the tumor cells,” Shipp remarked.
Twenty of the 23 patients had a measurable response to the treatment, with four achieving a complete response—in which no detectable tumor was left – and 16 having a partial response—in which their tumors shrank to less than half their original size. Six months after completing therapy, 86 percent of the patients were alive with continued responses. Most patients continue to do well a year after their treatment.
Side effects mirrored those that have occurred in tests of nivolumab in patients with solid tumors. About 20 percent experienced a serious treatment-related adverse effect but none was life-threatening.
The approach to immunotherapy embodied in nivolumab is a legacy of work at that began in the 1980s. HMS associate professor of medicine at Dana-Farber Gordon Freeman and Arlene Sharpe, the HMS George Fabyan Professor of Comparative Pathology, did some of the original research that resulted in the identification of PD-1 on T cells, as well as the PD-L1 and -L2 protein “ligands” on tumor cells.
“Their work was critical to our understanding that the increased presence of PD-L1 and -L2 on some tumor cells may allow those cells to escape attack from the immune system,” Shipp said.
Freeman and Sharpe’s work dovetailed with Shipp and her colleague’s own research into the genetic features of HL. “We found that Hodgkin lymphoma tumor cells frequently have an extra region of a specific chromosome that causes an increased production of the two ligands—PD-L1 and PD-L2—in the PD pathway,” Shipp said. “This characteristic genetic alteration suggested that inhibiting the PD-1 pathway might be particularly effective in this disease.”
Shipp and her co-authors offer two possible explanations for the high response rate to nivolumab in this study. One has to do with the sparse nature of HL. “Hodgkin lymphoma is unusual among cancers in that it consists of a small number of tumor cells in a sea of inflammatory cells and immune system cells, including T cells that don’t work very effectively.” Shipp observed. “Activating those T cells may create a very strong response to a relatively small number of cancer cells.” Another related possibility is the genetic trait that causes HL cells to produce an abundance of PD-L1 and PD-L2 ligands makes the tumor particularly vulnerable to PD-1 blockade.
For the investigators involved in the research, the results, though obtained in a relatively small, phase 1 trial, are compelling. “For someone like myself, in this kind of work, this is the kind of result that you get to see once in your career,” said study co-senior author Philippe Armand, HMS associate professor of medicine and medical oncologist in the Hematologic Oncology Treatment Center at Dana-Farber.
The study was supported by Bristol Myers Squibb, National Institutes of Health grants R01CA161026, U54CA163125 and P01AI056299 and with support from the Miller Family Fund to the Shipp laboratory.
Adapted from a Dana-Farber news release.
An analysis of circulating tumor cells (CTCs) in a mouse model of pancreatic cancer has identified distinct patterns of gene expression in several groups of these cells, including significant differences from the primary tumor that may contribute to their ability to spread. In a paper published in Cell Reports, Harvard Medical School investigators at the Massachusetts General Hospital Cancer Center pinpointed several different classes of pancreatic CTCs and found unexpected factors that may prove to be targets for improved treatment of the deadly tumor.
“Our ability to combine a novel microfluidic CTC isolation device, developed here at Mass General, with single-cell RNA sequencing has given us new biological insights into these cells and revealed novel avenues to try to block the spread of cancer,” said lead author David T. Ting, HMS assistant professor of medicine at Mass General.
Pancreatic cancer is among the most deadly of tumors because it spreads rapidly via CTCs carried in the bloodstream. The earliest technologies for isolating CTCs from blood samples relied on interactions with known tumor-specific marker proteins, potentially missing cells that did not express those particular markers.
The device used in the current study, called the CTC-iChip, enables the isolation of all CTCs in a blood sample, regardless of the proteins they express on their surface, by removing all other components. Since the CTCs collected are in solution, unlike with previous CTC capture devices, they are suitable for advanced RNA-sequencing techniques to reveal the gene expression patterns of each individual cell.
Using a well-known mouse model of pancreatic cancer, the researchers first isolated 168 single CTCs from the blood of five individual mice. Analysis of the RNA transcripts of each CTC revealed several different subsets of CTCs based on gene expression patterns that were different from each other and from the primary tumor.
The largest subset, which the authors call “classic CTCs,” was found to have elevated expression of a stem cell gene called Aldh1 a2. This subset of CTCs also had genes characteristic of two basic cell types—epithelial and mesenchymal. The transition between epithelial and mesenchymal cell types has been associated with tumor metastasis.
Another gene expressed by almost all classic CTCs, Igfbp5, is expressed only in primary tumors at locations where epithelial cancer cells interface with the supporting stromal cells that provide a nurturing microenvironment. This observation suggests that those regions may be the source of CTCs.
The research team was most surprised to observe that extracellular matrix (ECM) genes in general—usually expressed primarily in stromal, or connective tissue, cells—were highly expressed in all classic CTCs. Previous studies have suggested that the establishment of metastases depends on the appropriate cellular microenvironment—the “soil” in which CTCs can plant themselves as “seeds”—and that the expression of ECM genes is an important aspect of that environment. Expression of ECM genes by CTCs themselves suggests that the blood-borne cells may provide or help prepare their own “soil.”
Analysis of CTCs from blood samples of human patients with pancreatic, breast or prostate cancer also found elevated expression of several ECM genes. One particular gene, SPARC, was highly expressed in all pancreatic CTCs as well as in 31 percent of breast CTCs. Further experiments revealed that suppressing SPARC expression in human pancreatic cancer cells reduced their ability to migrate and invade tissue, and significantly fewer metastases were generated when SPARC-suppressed pancreatic tumors were implanted into a mouse model, supporting the protein’s role in a tumor’s metastatic potential.
“Given our limited therapeutic options for pancreatic cancer, understanding the role of the ECM in this tumor seems to be of great importance,” said Ting. “Much effort has been focused on targeting the microenvironment to improve the efficacy of chemotherapy, and data indicating that environmental stromal cells can enhance a tumor’s metastatic ability indicate that ECM proteins are important whether they are produced in stroma or within the tumor cells themselves. Now we need to investigate whether therapeutically targeting ECM can destroy both the tumor microenvironment and CTCs before they have a chance to metastasize.”
In 2011 Mass General entered a collaborative agreement with Janssen Diagnostics to establish a center of excellence in CTC research. Development of RNA in situ hybridization biomarkers was done with sponsored research support from Affymetrix Inc. Additional support for the current study includes a “Dream Team” grant from Stand Up to Cancer and grants from the Howard Hughes Medical Institute, Burroughs Wellcome Fund, National Institute of Biomedical Imaging and Bioengineering grant 5R01EB008047, and National Cancer Institute grant 2R01CA129933.
Adapted from a Mass General news release.
A collaboration between Harvard Medical School researchers at Brigham and Women’s Hospital and Dana-Farber Cancer Institute has utilized nanomedicine technologies to develop a drug-delivery system that can precisely target and attack cancer cells in the bone, as well as increase bone strength and volume to prevent bone cancer progression.
The study was published the week of June 30, 2014 in Proceedings of the National Academy of Sciences.
“Bone is a favorable microenvironment for the growth of cancer cells that migrate from tumors in distant organs of the body, such as breast, prostate and blood, during disease progression,” said Archana Swami, an HMS research fellow in anesthesia at the BWH Laboratory of Nanomedicine and Biomaterials and co-lead study author.
“We engineered and tested a bone-targeted nanoparticle system to selectively target the bone microenvironment and release a therapeutic drug in a spatiotemporally controlled manner, leading to bone microenvironment remodeling and prevention of disease progression,” Swami said.
“There are limited treatment options for bone cancers,” added Michaela Reagan, an HMS research fellow in Medicine at DFCI Center for Hematologic Oncology and co-lead study author.
“Our engineered targeted therapies manipulate the tumor cells in the bone and the surrounding microenvironment to effectively prevent cancer from spreading in bone with minimal off-target effects,” Reagan said.
The scientists developed stealth nanoparticles made of a combination of clinically validated biodegradable polymers and alendronate, a clinically validated therapeutic agent, which belongs to the bisphosphonate class of drugs.
Bisphosphonates bind to calcium. The largest store of calcium in the human body is in bones, so bisphosphonates accumulate in high concentration in bones.
By decorating the surface of the nanoparticles with alendronate, the nanoparticles could home to bone tissue to deliver drugs that are encapsulated within the nanoparticles and kill tumor cells, as well as stimulate healthy bone tissue growth.
Furthermore, bisphosphonates are commonly utilized during the treatment course of cancers with bone metastasis, and thus alendronate plays a dual role in the context of these targeted nanoparticles.
The scientists tested their drug-toting nanoparticles in mice with multiple myeloma, a type of bone cancer. The mice were first pre-treated with nanoparticles loaded with the anti-cancer drug, bortezomib, before being injected with myeloma cells.
Slower myeloma growth
The treatment resulted in slower myeloma growth and prolonged survival. Moreover, the researchers also observed that bortezomib, as a pre-treatment regimen, changed the make-up of bone, enhancing its strength and volume.
“These findings suggest that bone-targeted nanoparticle anti-cancer therapies offers a novel way to deliver a concentrated amount of drug in a controlled and target-specific manner to prevent tumor progression in multiple myeloma,” said Omid Farokhzad, HMS associate professor of anesthesia and director of the BWH Laboratory of Nanomedicine and Biomaterials, and co-senior study author.
“This approach may prove useful in treatment of incidence of bone metastasis, common in 60 to 80 percent of cancer patients and for treatment of early stages of multiple myeloma,” Farokhzad said.
“This study provides the proof of concept that targeting the bone marrow niche can prevent or delay bone metastasis," added Irene Ghobrial, HMS associate professor of medicine at DFCI Center for Hematologic Oncology and co-senior study author.
"This work will pave the way for the development of innovative clinical trials in patients with myeloma to prevent progression from early precursor stages or in patients with breast, prostate or lung cancer who are at high-risk to develop bone metastasis,” she said.
This research was supported by the United States Department of Defense (W81XWH-13-1-0390), National Institutes of Health (CA151884, CA133799), Movember-PCF Challenge Award, David Koch-Prostate Cancer Foundation Award in Nanotherapeutics, and a Friends of the Farber Grant.
It’s not cancer, but it grows and spreads to distant organs. It’s not malignant, but women die when it destroys their lungs. It has no cure, but scientists, physicians and patients are converging to change that.
Lymphangioleiomyomatosis — LAM for short — is a rare disease in which abnormal, smooth muscle-like cells grow out of control, usually in the kidney, lymph nodes and lungs. It develops almost exclusively in women during their childbearing years.
At first, they might feel shortness of breath, which is sometimes confused with asthma and sometimes caused by a collapsed lung. Some women find out they have the disease when CT scans their doctors order for other reasons reveal LAM’s telltale cysts, invisible on ordinary chest X rays but sprinkled throughout their lungs.
LAM is in many ways a mystery defined by what it is not. But the story of LAM has evolved rapidly over the past 15 years. That momentum accelerated when a team led by a physician-scientist now at Harvard Medical School identified the genetic mutation at its core and then developed a model that could be used to study it. Their work allowed the molecular machinery to be nailed down by scientists across the world.
A vibrant community of researchers has grown at HMS and the LAM Center at Brigham and Women’s Hospital. They in turn are linked to other research labs and hospital clinics around the country united by the LAM Foundation, a driving force in patient support and research funding.
“Our lab was in the right place at the right time to really start to make new contributions to understanding the pathology of LAM and the evolution of the disease,” said John Blenis, HMS professor of cell biology. “It turns out that two molecular pathways we’ve been studying for 20-plus years converge to contribute to LAM.”
At a meeting 15 years ago convened by the father of a patient, Blenis learned about the challenge from, among others, Elizabeth Henske, HMS professor of medicine at Brigham and Women’s and director of the LAM Center. An oncologist, Henske worked on tuberous sclerosis during her postdoctoral training. Tuberous sclerosis can cause LAM and severe cognitive problems, as well as unusual kidney tumors filled with blood vessels, smooth muscle tissue and fat cells. She had been puzzled because similar tumors occur in women with LAM.
Henske has devoted her career to solving the mystery of LAM. While a rare disease, new ways to treat it may have implications for more common disorders.
“These are tumor-like cells, but they’re not malignant-appearing. So it seems like it should be easier to fix than a cancer. And it affects almost only women, so there may be a hormonal element. It just seemed like something we ought to be able to figure out,” Henske said. “That’s what hooked me in and that’s still what I think about all the time.”
Madeline Nolan learned about LAM at the end of a months-long series of referrals to figure out why she was so fatigued and why her bloodwork looked so odd. A hematologist had sent her to a pulmonologist after ruling out a blood disorder. She was 47 years old, she taught phys. ed., and she was shocked.
“I don’t smoke, I’m not around toxic chemicals, how do I have a lung disease?” Nolan recalls wondering.
Nolan’s pulmonologist couldn’t tell her in 1999 why she had a disease that damages her lungs. With tears in his eyes, he told her she might have three or four years before needing a double-lung transplant.
She hasn’t gone down that road, although she now uses oxygen and takes a drug that has stalled her disease’s progression. Her story mirrors the mystery that LAM still is today.
Now teaching health to high schoolers in Waterbury, Conn., Nolan jokes that the pimples— a side effect from her medication—make her fit in with her students. She talks warmly about other LAM patients, who get together around their appointments at the LAM clinic in Boston as well at other events throughout New England. Her mission is to promote awareness, support patients and push research as the New England liaison for the LAM Foundation.
“I want to do everything I can to make a cure happen faster,” she said. “I want to do this for the next 20-year-old who gets diagnosed.”
Tuberous sclerosis and LAM share more than idiosyncratic kidney tumors. Tuberous sclerosis is an inherited disease caused by mutations in genes called TSC1 and TSC2, which patients carry in every cell in their bodies. About 40 percent of girls with tuberous sclerosis also go on to develop LAM in their childbearing years. Women without tuberous sclerosis have a sporadic form of LAM: They carry mutations only in the TSC2 gene and only in their tumor cells.
Henske participated in the cloning of the TSC1 gene in 1999, and she was the one who discovered in 2000 (while working at Fox Chase Cancer Center) that LAM is caused by mutations in TSC2.
At that point, no one knew what TSC2 did. But in what Henske calls a moment almost too good to be true, scientists studying flies to find genes that enlarge cells in the eye discovered that TSC genes control cell size. Other scientists determined that these genes regulate an enzyme called mTOR, a key player in a series of molecular events involved in cancer and one of two major signaling systems studied in the Blenis lab since the early 1990s.
The link between TSC and mTOR led quickly to a clinical trial showing that rapamycin, a natural product that blocks the mTOR pathway (and gives the enzyme its name: mammalian target of rapamycin complex), could shrink kidney tumors in women with LAM. A later clinical trial found that rapamycin could also stabilize lung function in women with LAM. It’s not a cure—the disease continues to destroy the lungs if a woman stops taking the drug—but it stalls further lung damage.
“This is a really beautiful example of pure science,” Henske said. “Looking for regulators of cell size in the fly eye led in five years to the completion of a trial in a disease that is just devastating. You can’t imagine what it’s like for a young woman who’s already on oxygen.”
Meeting LAM face to face
Henske thinks about those women all the time. To get to her desk, she walks by a quilt hung outside her office honoring women with LAM. Squares depict lives stitched together at LAM Foundation events where scientists, women with LAM and their families all rub shoulders and form bonds. Physicians and researchers give presentations at annual meetings, but patients speak too, telling their stories and touching the hearts of basic scientists who don’t always see the people whose lives they hope to help.
Henske’s colleague in a nearby office, Jane Yu, proudly wears a new navy blue fleece top embroidered with the LAM Center’s name, a gift from Henske on the center’s fifth anniversary. HMS assistant professor of medicine at Brigham and Women’s, Yu led pioneering research that created the first animal model that closely recapitulates how LAM metastasizes under the influence of estrogen. She later showed that a drug can block LAM cells from spreading to the lung by inhibiting estrogen, confirming the hormone’s role in the disease.
Yu finds the LAM meetings unusual—and inspiring.
“Once you are there, you are family: Your sisters are there, your aunts are there. I wouldn’t say grandmothers—yet,” she said. “You feel that you are one of them and you want to help. They never give up, and every year I think we should try harder.”
Blenis said by going to LAM meetings, he became more interested in researching the disease. “I get very emotional when I go to these meetings, and I think all my postdocs who have ever gone have felt the same way.”
Xiaoxiao Gu, a postdoc in the Blenis lab, is also a LAM Foundation fellow. She had two questions she wanted to answer when she started her three-year fellowship in 2011. “Because the hypothesis is that these kidney tumor cells are migrating to the lung in the presence of estrogen, how can I test estrogen responsiveness in those cells? And why is an inhibitor like rapamycin not enough to block disease progression?”
In a PNAS paper published in September 2013, Gu and Blenis offered an explanation: The two pathways that the Blenis lab has played a major role in defining actually converge in LAM in a way that requires their precise spatial and temporal regulation to generate the disease.
It takes more than the mTOR pathway—the set of molecular activities that rapamycin can inhibit, thereby blocking runaway cell metabolism and growth. It also takes a signaling pathway involved in the development of cancer: a chain of communicating proteins called the estrogen receptor-MAP (ERK-MAP) kinase pathway that transmits signals from the cell surface to DNA in the nucleus of the cell.
The team built on work by Yu and Henske showing that estrogen increases the migration of cells with TSC2 mutations via the MAP kinase pathway. Gu and Blenis found that estrogen signaling in the ERK-MAP kinase pathway is active in LAM cells, establishing that estrogen collaborates with inappropriate regulation of mTOR, converging to generate disease.
The ERK-MAP kinase pathway, activated by estrogen, starts a process that makes cells more likely to migrate, invade and survive. The mTOR pathway provides the energy and building blocks to support LAM cell growth and to enhance the estrogen-stimulated events.
“It’s this amazing integration of different signals at different times that contributes to disease progression,” Blenis said. “By discovering how these two pathways converge, it also suggests possible therapeutic options.”
The MAP kinase pathway has already been targeted by pharmaceutical companies for other reasons, Blenis said.
“We’re defining mechanistically how these pathways converge and verifying the choice of using an estrogen inhibitor in combination with mTOR inhibitors such as rapamycin,” Blenis said. “One drug may have some effects, but the combination of the two is going to be significantly greater at treating the disease.”
Blenis, Henske, Yu and Gu all profess optimism with the field’s rapid progress, confident that what they learn about LAM may be applied to other diseases, including cancer.
“The tuberous sclerosis genes are in the center of the universe in terms of how a cell regulates almost everything it does, from metabolism to growth to its decision to divide. Everything depends on mTOR,” Henske said. “Many of us who study rare diseases have the very strong belief that the way you figure out a common disease is almost always by starting with a rare disease.”
Knowing the one faulty gene where a rare disease starts is much better than looking at 10 or 100 or 1,000 genes involved in a common disease, Henske said. “That’s kind of like looking at a crashed car and trying to figure out if it was the steering wheel or the brakes.”
In the Blenis lab, LAM is directly linked to his work on breast cancer metastasis while remaining significant in its own right. LAM patients’ pull on the scientists is strong.
“Clinician-scientists have more of a direct impact on patients,” Blenis said. “For a basic scientist, this is the whole reason to get into the field: We hope our work will have an impact down the road.”
Madeline Nolan and her LAM sisters are waiting.
“We’ve come such a long way in a short time.”
When a person suffers a broken bone, treatment may call for the surgeon to insert screws and plates to help bond the broken sections and enable the fracture to heal. These “fixation devices” are usually made of metal alloys.
These metal devices have disadvantages: Because they are stiff and unyielding, they can cause stress to underlying bone. They also pose an increased risk of infection and poor wound healing. In some cases, the metal implants must be removed following fracture healing, necessitating a second surgery. Resorbable fixation devices, made of synthetic polymers, avoid some of these problems but may pose a risk of inflammatory reactions and are difficult to implant.
Now, using pure silk protein derived from silkworm cocoons, a team of investigators from Harvard Medical School, Beth Israel Deaconess Medical Center and Tufts University School of Engineering has developed surgical plates and screws that may offer improved bone remodeling following injury. Equally important, the devices can also be absorbed by the body over time, eliminating the need for surgical removal.
The findings, demonstrated in vitro and in a rodent model, are described in the March 4 issue of Nature Communications.
“Unlike metal, the composition of silk protein may be similar to bone composition,” said co-senior author Samuel Lin, HMS associate professor of surgery in the Division of Plastic and Reconstructive Surgery at Beth Israel Deaconess. “Silk materials are extremely robust. They maintain structural stability under very high temperatures and withstand other extreme conditions, and they can be readily sterilized.”
Lin worked with co-senior author and Tufts chair of biomedical engineering David Kaplan. “One of the other big advantages of silk is that it can stabilize and deliver bioactive components, so that plates and screws made of silk could actually deliver antibiotics to prevent infection, pharmaceuticals to enhance bone regrowth and other therapeutics to support healing,” said Kaplan.
First author Gabriel Perrone, of the Department of Biomedical Engineering at Tufts, used silk protein obtained from Bombyx mori silkworm cocoons to form the surgical plates and screws. Produced from the glands of the silkworm, the silk protein is folded in complex ways that give it unique properties of both exceptional strength and versatility.
To test the new devices, the investigators implanted a total of 28 silk-based screws in six laboratory rats. Insertion of screws was straightforward and assessments were then conducted at four weeks and eight weeks, post-implantation.
“No screws failed during implantation,” said Perrone, explaining that because silk is slow to swell, the new devices maintained their mechanical integrity even when coming into contact with fluids and surrounding tissue during surgery. The outcomes suggest that the use of silk plates and screws can spare patients the complications that can develop when metal or synthetic polymer devices come into contact with fluids.
“Having a resorbable, long-lasting plate and screw system has potentially huge applications,” said Lin. While the initial aim is to use silk-based screws to treat facial injuries, which occur at a rate of several hundred thousand each year, the devices have potential for the treatment of a variety of different types of bone fractures.
“Because the silk screws are inherently radiolucent (not seen on X-ray) it may be easier for the surgeon to see how the fracture is progressing during the post-op period, without the impediment of metal devices,” added Lin. “And having an effective system in which screws and plates ‘melt away’ once the fracture is healed may be of enormous benefit. We’re extremely excited to continue this work in larger animal models and ultimately in human clinical trials.”
This research was supported by the National Institutes of Health (EB002520).
Adapted from a Beth Israel Deaconess news release.
In order to better combat infectious disease, Harvard Medical School is creating three Centers of Excellence for Translational Research: one in tuberculosis, one in bacteriology and one in virology.
Three five-year grants totaling up to $15 million per year from the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, will allow HMS researchers to move discoveries about TB and emerging infections closer to applications in diagnosis, treatment and prevention.
Improving diagnosis, fighting drug resistance
The TB center will focus on improving diagnostics, especially in children, and on combatting drug resistance. Megan Murray, HMS professor of global health and social medicine, will lead the center.
“The TB epidemic is still fueled by the fact that people are diagnosed relatively late in the course of their disease and a lot of transmission happens before diagnosis,” said Murray, who is also HMS associate professor of medicine at Brigham and Women’s Hospital and director of research at Partners in Health. “There’s no single therapy for TB, so there’s a big need to know which drugs people are resistant to.”
The TB center will build on previous work that used whole-genome sequencing to identify genetic mutations associated with drug resistance. Using bioinformatics and evolutionary techniques, they will study some 1,500 TB strains collected from an ongoing clinical research study in Peru to characterize resistance mechanisms. The scientists will correlate what they find with a measure called quantitative drug resistance, or the specific amount of a drug to which a strain becomes resistant.
Another project in the center, to be led by Eric Rubin, professor of immunology and infectious disease at the Harvard School of Public Health, will apply functional genomics to the mutations found by sequencing to see if the mutations confer drug resistance.
The TB center will work with an industry partner, Akonni Biosystems, to develop a diagnostic tool to be used in the field. Led by Chief Scientific Officer Darrell Chandler and Director of Engineering Christopher Cooney, the molecular diagnostics company will optimize a microarray for TB to test mutations associated with drug resistance.
The TB center’s fourth of four projects may be its most ambitious, Murray said. Inspired in part by techniques used to interpret archeological DNA in Neanderthal samples, the scientists hope to capture fragments of TB’s genetic material from children. The standard TB test looks at sputum, but children can rarely cough up a useful sample. What if clinicians could examine bits of DNA in blood or urine and do other diagnostic tests as well?
“In the Neanderthal project, the challenge has been to take very degraded mammalian DNA mixed with bacterial DNA,” she said. “In our case, we’ve got the other problem: We try to pull microbial DNA mixed with human DNA in urine. Can we sequence that?”
Searching for new defenses
The two new centers in bacteriology and virology will build on the success of the New England Regional Center for Excellence, a regional research hub established at HMS after anthrax attacks in 2001 heightened national concerns about biological threats. Dennis Kasper, the HMS William Ellery Channing Professor of Medicine at Brigham and Women’s Hospital and professor of microbiology and immunobiology at HMS, is the principal investigator on the NERCE grant.
HMS researchers will continue to focus on pathogens and diseases for which no vaccines or therapies exist—and microbes that resist current drugs. These include the bacterium Francisella tularensis, considered a potential agent of bioterrorism, as well as dengue fever, a significant disease in much of the world, which is now creeping into Florida.
“In NERCE, there was a lot of basic research on biodefense pathogens and later on emerging infectious disease pathogens,” added Kasper. “These new centers are now here to take those discoveries to the next step: translation.” Kasper will head the bacteriology CETR, a program that will focus on tackling the cell envelope that surrounds bacteria in such microbes as burkholderia, brucella, Vibrio cholerae and methicillin-resistant Staphylococcus aureus.
The virology CETR will be led by Sean Whelan, professor of microbiology and immunobiology and associate head of the Harvard Program in Virology. “This really is an opportunity to make a significant impact in understanding the entry mechanisms of emerging viral pathogens,” Whelan said. “We’re going to learn new biology and understand how some small molecules block viral entry.”
Viral entry is a target of our natural antibody response to viral infection, a step in the viral replication cycle that has not been well exploited by synthetic inhibitors.
One virology project involves small molecules that inhibit dengue virus replication and another concerns small molecules that block the Ebola virus from getting into cells. A third effort will search for cellular molecules that many viruses use to reach and enter cells. A fourth project will explore how viruses move from one cellular compartment to another before infecting cells with their genes.
New science for emerging threats
“We are looking at a very broad spectrum of emerging viruses that are threats to human health and potential biodefense agents, asking what molecules and cellular factors are needed to get into cells,” Whelan said. “We’re hoping that we will identify both pathogen-specific host factors as well as those that are shared among different viruses.”
In a similar vein, scientists in the bacteriology CETR hope to find common weaknesses to exploit.
“A lot of antibiotic research addresses enzymes or proteins that are involved in the synthesis of the cell wall of bacteria. There are key proteins that are very similar between different bacteria that could potentially serve as targets,” Kasper said. “Vaccines tend to be organism-specific, but we’re developing platforms that can be applied to any organism for which you want to develop a vaccine.”
“For many years it was sufficient for people to talk about their basic research as perhaps leading to a drug or to a vaccine someday,” said Gerald Beltz, administrative director for the two centers in the Department of Microbiology and Immunobiology. “Now it has to be much more direct, and I think these centers are part of that.”
HMS investigators within the bacteriology CETR are John Mekalanos, the Adele Lehman Professor of Microbiology and Molecular Genetics and head of the department of microbiology and immunobiology; Suzanne Walker and Stephen Lory, both professors of microbiology and immunobiology; Thomas Bernhardt and David Rudner, associate professors of microbiology and immunobiology; and Daniel Kahne, professor of biological chemistry and molecular pharmacology.
Virology CETR investigators at HMS include James Cunningham, associate professor of medicine (microbiology and immunobiology) at Brigham and Women’s Hospital; Stephen Harrison, the Giovanni Armenise-Harvard Professor of Basic Biomedical Science; Tomas Kirchhausen, professor of cell biology; Priscilla Yang, associate professor of microbiology and immunobiology; and Nathanael Gray, professor of biological chemistry and molecular pharmacology.
Other researchers involved in the TB center include Max Salfinger, lab director of mycobacteriology and pharmacokinetics at National Jewish Health in Denver, who will lead quantitative genomics project. In the later years of the TB grant, scientists will work with Ann Goldfeld, HMS professor of medicine at Boston Children’s Hospital, who heads a clinical research site in Cambodia. Louise Ivers, HMS associate professor of Global Health and Social Medicine and associate professor of medicine at Brigham and Women’s Hospital, will lead similar clinical research in children in Haiti.
James Sacchettini, professor of biochemistry and biophysics and of chemistry at Texas A&M University, and Thomas Ioerger, associate professor of computer science at Texas A&M, will lead gene sequencing.