Research Area: Human Disease

A Vision for Science

Clare Harding's microscope image of Toxoplasma gondii parasites is one of this year's winners at the Koch Institute Image Awards

Nicole Giese Rura
March 9, 2018

Scientists use a variety of approaches to unravel the functions of organisms, cells, and even molecules, and some of these approaches produce images that are as stunning as they are informative.  Since 2011, the annual Koch Institute Image Awards, conducted by The Koch Institute for Integrative Cancer Research at MIT, has honored outstanding images created by life science and biomedical researchers in the MIT community.

This year, one of the winning pictures was created by Clare Harding, a postdoctoral researcher in the lab of Whitehead Institute Member and MIT assistant professor of biology, Sebastian Lourido. Harding and the other winners were lauded last night at a gala opening of the exhibit on the Koch building’s ground floor where the winning images will be on display for the coming year.

Whitehead has participated in this contest since its inception, with winning images by Gianluca De Rienzo (postdoctoral researcher in Whitehead Member Hazel Sive’s lab) in 2011, Rob Mathis (graduate student in Whitehead Member Piyush Gupta’s lab) in 2013, Daphne Superville (undergraduate student in Gupta’s lab) in 2015, Dexter Jin (graduate student in Gupta’s lab) in 2016, and Samuel A. LoCascio and Kutay Deniz Atabay (graduate students in Whitehead Member Peter Reddien’s lab) in 2017.

In Harding’s striking entry this year, each white and blue “petal” of the rosettes is a single-celled Toxoplasma gondii, the parasite that causes toxoplasmosis infection. This image was taken moments before the individual parasites comprising the daisy-like clusters would have triggered a massive, coordinated “egress”, which would destroy the host cell they had called home. The host’s nucleus is the large blue oblong jutting in from the upper left (host and parasite DNA are marked blue), and the red dapple marks a molecule in the host cell’s internal skeleton, called tubulin.

Toxoplasmosis infects about 25% of the world’s population and can cause serious disease in pregnant women, infants, and immunocompromised people. Not only is the Lourido lab’s work on T. gondii revealing important clues about this disease, but their research can also shed light on T. gondii’s close cousins on the evolutionary tree: Plasmodium spp., which cause malaria and contribute to more than a million deaths each year; and Cryptosporidium spp., which cause cryptosporidiosis, a gastrointestinal illness that can be fatal in those with a compromised immune system.

Harding’s research in the Lourido lab is focused on GAPM1a, a structural protein that forms a layer directly under T. gondii’s outer membrane and plays a similar architectural role in Plasmodium. This protein scaffold (marked as white in the image) is so vital that it is one of the first things established within daughter cells, which appear in the image as two small white spheres within some of the larger parasites. Parasites lacking the GAPM1a scaffold degrade into amorphous blobs that are unable to infect new host cells—a visual testament of how important this protein is to the parasites.

Light microscopy images like Harding’s are created by passing or reflecting light off of a sample and using one or more lenses to magnify the resulting representation. According to Wendy Salmon, the light microscopy specialist at Whitehead’s W.M. Keck Biological Imaging Facility and a two-time Koch Image Awards judge, all microscopy-based images are imperfect representations of the samples that they depict, because light microscopy is limited by the physics of the light shined on the sample and the glass that comprises the lenses. To push beyond the boundaries of physics and reveal the otherwise invisible, Harding employed two techniques: fluorescent markers and structured illumination microscopy.

Using a light microscope alone, the GAPM1a protein is indiscernible within T. gondii parasites.  But by genetically modifying the parasite to produce the GAPM1a protein fused to a green fluorescent protein, Harding could shine a particular wavelength of light onto the sample and cause the fluorescent protein to glow, thereby illuminating GAPM1a’s presence.

In addition to being able to identify the protein she is studying in a sample, Harding has the additional challenge that the parasites are so tiny—5 micrometers in length, or about 1/16th the width of a human hair—that they are beyond the resolution of light microscopy. In order to visualize the GAPM1a scaffold, Harding used a technique called structured illumination microscopy, which takes advantage of the properties of light in order to see things half the size of what is visible with a conventional light microscope. In this technique, the microscope casts a grid of light onto the specimen and takes images as the grid rotates. The resulting data from the images are processed using an algorithm that reconstructs the specimen’s appearance, enhancing its resolution.

Harding has been working with T. gondii for more than three years and microscopy has always played a major role in her work, but her appreciation for the science and art of microscopy has recently flourished.

“I like microscopy partly because it’s beautiful and partly because with a lot of other techniques, you need to interpret the data. With microscopy, you know what you’re looking at is right there,” says Harding, who is thrilled to have her work featured in the Koch Institute Public Galleries. “I definitely fell in love with microscopy right away. The first time I did it, I realized how much there is to a cell. Even just staining the DNA in a cell, suddenly you can see stars.”

Toxic proteins and type 2 diabetes

Whitehead Institute study in yeast illuminates the role of a molecular de-clogger in disease biology.

Nicole Davis | Whitehead Institute
March 8, 2018

Nearly half a billion people worldwide live with type 2 diabetes. Yet despite the disease’s sizeable and increasing impact, its precise causes remain murky. Current scientific thinking points to two key processes: insulin resistance, wherein cells develop ways of tuning out insulin’s signals, and the breakdown of beta cells, the specialized cells in the pancreas that produce insulin. The molecular bases for these activities, however, are largely unknown.

Now, a team of researchers based at Whitehead Institute is casting new light on the theory that abnormal protein deposits — similar to ones that emerge in neurodegenerative disorders such as Alzheimer’s disease — accumulate in and around beta cells and derail their normal function. The team’s findings, which appear in today’s advance online edition of the journal Cell, illuminate the function of a key protein, called Ste24, which unclogs the cellular machinery that helps shuttle proteins into compartments within the cell. The researchers believe that this unclogging action could be an important way to minimize or even prevent the protein deposits that damage precious beta cells in type 2 diabetes.

“We’ve created a new platform for identifying potential genetic and pharmaceutical targets that can help neutralize the toxic proteins that build up in patients with the disease,” says lead author Can Kayatekin. “In initial studies with this platform, we unveiled a very interesting target, Ste24, which has opened an important window on the biology of proteotoxicity in type 2 diabetes.”

Alongside the glucose-lowering hormone insulin, beta cells produce another protein, called IAPP (short for human islet amyloid polypeptide). As these two proteins mature inside the cells, they are bundled together and released within the same miniature packets, or vesicles. However, as its name suggests, IAPP is very amyloidogenic — that is, prone to forming large aggregates, which can pile up both within and outside of cells.

“What happens is that as demand for insulin increases, you get more and more IAPP production, and the more you make, the more likely it is to aggregate,” Kayatekin says. “So, the idea is that as you make more IAPP, it starts poisoning the very cells that are producing it.”

To further explore the molecular mechanisms of IAPP production and aggregation, Kayatekin harnessed a powerful paradigm established by his late mentor and supervisor Susan Lindquist, a Whitehead Institute member, MIT professor of biology, and HHMI Investigator who passed away in 2016. Her pioneering approach leverages the baker’s yeast Saccharomyces cerevisiae to create models of toxic proteins in order to probe, perturb, and expose their underlying biology.

Kayatekin and the study’s co-authors generated a yeast model that carries six tandem copies of IAPP. “In most of the neurodegenerative and protein aggregation diseases, the research has trended towards these kinds of smaller oligomers, which seem to be more capable of diffusing in the cell and are therefore likely to be more toxic,” he explains.

With their model of IAPP toxicity in hand, the researchers then turned to genetic techniques to identify yeast proteins that either enhance or ameliorate the effects of IAPP aggregation. Kayatekin and his team identified several intriguing finds, perhaps the most interesting one being a protease called Ste24. According to a 2016 study published in Cell by Maya Schuldiner’s laboratory, Ste24 can cleave proteins that clog translocons — the channels through which secreted proteins, including IAPP, must pass before they can be released. Much like liquid drain cleaners can clear household pipes of hair balls and other muck, Ste24 can remove proteins that get stuck as they venture through the cell’s inner straits. Indeed, Kayatekin finds that overexpressing Ste24 in his yeast model can help rescue some of the effects of IAPP deposits.

Notably, Ste24 is highly conserved through evolution — so much so that the human version, ZMPSTE24, can stand in for its yeast counterpart, the researchers found. This remarkable feature allowed the team to begin functionally dissecting how natural variation in the human protein might impact its unclogging function. By scouring different genetic variants in ZMPSTE24 identified with the help of the AMP T2D-GENES Consortium, they discovered versions whose function was impaired. Initial data suggests that some of these loss-of-function mutants may be more common in type 2 diabetes patients than those without the disease — suggesting that a less-than-robust declogger could possibly contribute to type 2 diabetes progression.

More work is needed to fully decipher the biology of Ste24, IAPP toxicity, and type 2 diabetes. Nevertheless, Kayatekin hopes that his innovative yeast model will prove to be as powerful a tool for illuminating the molecular underpinnings of disease as the ones that preceded it.

Funding for this work was provided by Whitehead Institute, the Picower Institute at MIT, the University of Texas, M.D. Anderson Center, the Howard Hughes Medical Institute, the Glenn Foundation for Medical Research, the Eleanor Schwartz Charitable Foundation, the Edward N. and Della L. Thome Foundation, the JPB Foundation, the Robert A. and Renee E. Belfer Foundation, the National Institutes of Health, the Canadian Institute of Health Research, and the U.S. Department of Defense. The researchers received additional support from the American Italian Cancer Foundation, the American Parkinson’s Disease Foundation, and the Helen Hay Whitney Foundation.

New study solves an arthritis drug mystery

MIT biological engineers discover why a promising drug failed in clinical trials.

Anne Trafton | MIT News Office
March 6, 2018

Pharmaceutical companies once considered a protein called p38 a very attractive target for treating rheumatoid arthritis. Arthritis patients usually have elevated activity of this inflammation-producing protein, and in lab studies p38 inhibitors appeared to soothe inflammation. However, these drugs failed in several clinical trials.

A new study from MIT sheds light on just why these drugs did not work for arthritis. By untangling the complex interactions between different cell pathways involved in inflammation, the researchers discovered that shutting off p38 triggers other inflammatory pathways.

The findings demonstrate the importance of studying a potential drug’s impact on complex cellular systems, says Doug Lauffenburger, head of MIT’s Department of Biological Engineering and the senior author of the study. It’s also important to do these studies under environmental conditions that match those found in diseased tissue, he adds.

“You’ve got to make sure you understand the complexity of the intracellular networks, and beyond that, you need to think about the environment you put the cells in,” Lauffenburger says. “It’s easy to get different results in different contexts, so you need to study them under many different conditions.”

Former MIT postdoc Doug Jones is the lead author of the paper, which appears in the March 6 issue of Science Signaling.

A promising target

Rheumatoid arthritis, which afflicts more than 1 million Americans, is an autoimmune disorder that produces swollen and painful joints, primarily affecting the wrists and hands. This pain results from inflammation in the lining of the joints. Cells called synovial fibroblasts, which typically provide structural support for the joint lining, promote the inflammation and swelling in arthritic conditions.

Several years ago, scientists seeking new treatments for arthritis discovered that synovial fibroblasts from arthritis patients had very high levels of p38, and many pharmaceutical companies began working on p38 inhibitors. “The activity of this pathway was so strong that people tended to think that it was the best one to inhibit,” Lauffenburger says.

Despite their promise, p38 inhibitors failed in phase II clinical trials run by at least eight pharmaceutical companies. One of those companies, Boehringer Ingelheim, asked Lauffenburger to help them figure out why. Lauffenburger’s lab focuses on systems biology, a field that involves measuring the interactions of many cell components and then performing computational modeling of those measurements to predict cell behavior.

The researchers’ analysis revealed that the inflammatory pathway controlled by p38 interacts with several other pathways that can cause inflammation. These pathways, known collectively as stress pathways, produce inflammatory cytokines in response to events such as infection or injury.

The MIT team found that when p38 is extremely elevated, it suppresses the activity of these other inflammatory pathways. Therefore, when it gets turned off, the brake on the other pathways is released. Under these circumstances, inflammation remains high — the difference is that now it is controlled by other stress pathways.

“This is an insightful paper on redundancy in signaling and the need to understand compensatory mechanisms before spending billions on drug development. In that sense, it is a far more important insight than ‘just’ p38 inhibitors, and it makes clear again that animal efficacy models have severe limitations as tools to predict human efficacy,” says David De Graaf, CEO and president of Syntimmune, who was not involved in the research. “This paper outlines one very thoughtful and generic approach to answer complex questions about efficacy in ex vivo human model systems.”

Environment matters

Why was the MIT team able to see this phenomenon when others had not? Lauffenburger says one key is the environment in which the synovial fibroblast cells were studied.

Normally, cells studied in the lab are grown in a culture medium that offers them nutrients and molecules called growth factors, which keep the cells alive and proliferating. However, the MIT team found that under these conditions, a pro-growth pathway called MEK actually keeps p38 levels lower than in cells under stress. Because p38 is not as high, it doesn’t inhibit the other stress pathways as strongly, so when the cells are exposed to p38 inhibitors, the other pathways don’t soar into action and overall inflammation goes down.

“It looks like p38 inhibitors work well, if cells are in these growth factor environments,” Lauffenburger says.

However, the MIT team found that synovial fluid from arthritis patients is not a pro-growth environment but is full of inflammatory cytokines. They then decided to expose synovial fibroblasts taken from patients with arthritis and from healthy individuals to this inflammatory environment. In both healthy and diseased cells, p38 levels skyrocketed, producing more inflammation and shutting off other stress pathways.

One question still to be answered is whether p38 inhibitors could work against other diseases such as cancer, in which the cells targeted would likely be in a pro-growth environment. They are also being considered as potential treatments for other inflammatory diseases such as multiple sclerosis and Alzheimer’s. Lauffenburger says that their success will likely depend on what kind of environment the affected cells are in.

“A p38 inhibitor could work; you just have to know what the context is that the target cells are in. If you have the same kind of inflammatory cytokines there, then you might encounter the same problem” seen in arthritis, he says.

It’s also possible that p38 inhibitors could work against arthritis or other drugs if given along with drugs that shut off other stress pathways, but more research would be needed to investigate that possibility, Lauffenburger says.

The research was funded by the National Institutes of Health, the Army Research Office, and Boehringer Ingelheim Inc. The project was undertaken in collaboration with Professor Peter Sorger at Harvard Medical School; Brian Joughin at MIT and Anne Jenney at Harvard were also significantly involved in the work.

Tracking tumorigenesis

Elizabeth Li ’18 has worked in three cancer-related labs over the past six years, and one day intends to start her own.

Raleigh McElvery
March 6, 2018

Senior Elizabeth Li recreates miniature organs — lungs and intestines — outside the body. She does so in order to study cancer progression in both environments, and over the past six years has worked in three separate cancer-focused labs: two at MIT and another beginning her junior year of high school. One day, she aims to run her own.

“I’ve been into math and science ever since I was little,” she explains, “but in third grade I met a friend who was pretty important to me. She was diagnosed with a very malignant form of brain cancer and ended up dying from it. From that point on — even though I was still very young — I knew I wanted to do cancer research.”

In 9th grade, Li began at the School for Science and Math at Vanderbilt, a joint program between the university and Metro Nashville Public Schools. “I got to skip school once a week to learn research techniques, and had the opportunity to join Andries Zijlstra’s lab my junior year,” she recalls. “I’m actually still part of that group, and I’ve been working on the same project related to cancer metastasis for six years now.”

When it came time to select a university, Li was torn between Vanderbilt — where she was already performing research — and MIT, which she describes as “the place of opportunity.” She was ultimately swayed by MIT’s vast array of research areas, and fully sold after an overnight to preview the undergraduate culture.

Li opted for Course 7 in order to continue doing cancer research, and joined Omer Yilmaz’s lab in 2015 to investigate intestinal tumorigenesis. Here, she spent most of her time doing organoid work, studying the progression of colorectal cancer in miniaturized and simplified versions of the intestine. Li removed individual intestinal stem cells — or sometimes the entire “crypts” in which they reside — and grew them inside a 3D gel. This environment allowed the cells to differentiate and interact as they would in the colon, rather than growing on a flat, plastic dish.

Li and other members of the Yilmaz lab watched these cells multiply, observing their shape and the regeneration process. Li’s method of assessment varied depending on the research question: on some days, she stained the cells for proliferation markers, and on others she exposed them to different metabolites or drugs to see how the cells responded.

“On a typical day, I would come in during the morning between classes, and again in the afternoons and evenings,” she says. “The experiments differed, but we tended to do a lot of genetic manipulation. We’d make plasmids, CRISPR-Cas9 knockouts, or test for gene and protein expression using qPCR and Western blots.”

After two years, Li’s primary mentor finished her postdoctoral training, and Li transitioned to Jackie Lees’ lab at the beginning of her senior year. Li is now working with a fellow undergraduate on an independent project, centered on the enzyme protein arginine methyltransferase 5 (PRMT5).

PRMT5 catalyzes the transfer of methyl groups to the amino acid arginine in certain proteins, modifying their function. The enzyme also plays a key role in regulating gene splicing, the process by which segments of pre-mRNA are removed — changing the genetic code so that multiple genes can be encoded by the same initial transcript.

The Lees lab is interested in PRMT5 because it affects glioblastoma formation, the most common form of adult brain malignancy. As Li explains, when PRMT5 expression increases, so does tumor formation. Since there are still relatively few therapeutic options to treat glioblastomas, she’s hoping to use small molecules to inhibit PRMT5 expression and thus impede tumor initiation and progression.

“We’re considering using nanoparticles to deliver them,” she says, “and in doing so, hoping to gain a better understanding of how PRMT5 inhibition might impact cancer progression and tumorigenesis.”

Li is testing one small molecule PRMT5 inhibitor in lung organoids and several 2D cell lines — determining how sensitive the cells are and if the organoids will form, to gauge whether a tumor would still develop in the presence of the drug. “Depending on when you add the drug, you can test different aspects of tumorigenesis,” she explains.

She’s also split the past four years between the Biology Undergraduate Student Association (of which she was faculty liaison, outreach chair, and then co-president), the MIT Pre-Medical Society, MIT Lion Dance, Asian Dance Team, Wind Ensemble, and Improv-a-Do! She’s also heavily involved in DynaMIT, which organizes an annual, week-long science program for economically disadvantaged middle school students.

“There are a lot of extracurriculars to do,” Li admits. “But it’s pretty easy to get involved in the MIT community and still stay on top of your coursework, if you keep it to four or five classes per semester. It’s worked out for me so far — I’m still alive and happy and have time for eating, sleeping, and friends.”

As Li applies to MD-PhD programs, she hopes one day to practice medicine (perhaps pediatric oncology) while running her own lab.

“My advice for incoming MIT undergrads would be to remember to have fun,” she says. “You only have four years, so take advantage of your time here: hang out with your friends, take the classes you want to take, and do things that you enjoy. Hopefully most of those activities will be one and the same.”

Photo credit: Raleigh McElvery
Probing a critical player in cancer growth

Alissandra Hillis ’18 has spent all four years at MIT in the same cancer metabolism lab, deciphering the basic science behind pancreatic cancer.

Raleigh McElvery
February 19, 2018

Senior Alissandra Hillis attributes her appetite for the basic sciences to her craving for fundamental knowledge. She’s spent her four years at MIT in the same lab, committed to unraveling the molecular mechanics of pancreatic cancer — the fourth leading cause of cancer death for both men and women, given that symptoms do not often appear until the disease is quite advanced.

“I was always very curious growing up,” she says. “I taught myself how to read at a very young age, just because I wanted to know about things and how they worked. But I didn’t become interested in biology and chemistry specifically until I came to MIT and started taking my General Institute Requirements.”

In doing so, Hillis became enthralled by the prospect of breaking down life into its most fundamental, biological units to decipher cellular function and disease. Originally a Course 7 major with a chemistry minor, she declared Course 5-7 (Chemistry and Biology) as soon as it became available in the fall of 2017 — applying her study of biochemistry and cell metabolism to cancer research.

“When I was quite young, my grandfather was diagnosed with stomach cancer, and ended up having almost three quarters of his stomach removed,” she says. “I was too little to really understand the severity of the situation, but as soon as I came to MIT I started to wonder what was going on at a cellular level. Most people today know someone who is fighting cancer, and yet we’re still lacking effective treatments for its most severe forms.”

Hillis joined Matthew Vander Heiden’s cancer metabolism lab the first semester of her freshman year, and has been there ever since.

“Professor Vander Heiden does an excellent job of tailoring the research project to the individual, and there is no hierarchy among lab members,” she says. “I really liked it from the onset, so I stayed.”

For nearly two years, Hillis has been investigating the role of one enzyme, pyruvate kinase muscle isozyme M2 (PKM2), in pancreatic cancer. PKM2 is responsible for catalyzing the final step in glycolysis, which is required to create the energy that fuels cells. Glycolysis is also important in tumor metastasis and growth, since cancer cells demand energy in order to proliferate.

Cancer cells often preferentially express PKM2 over other types of pyruvate kinases such as PKM1. This spurred William Israelsen PhD ’14, a former graduate student in the Vander Heiden lab working in breast cancer models, to delete the PKM2 gene and see what happened. Since PKM2 is critical for glycolysis, and cancer cells require energy to proliferate, he anticipated that removing PKM2 would hinder energy production and thus disrupt tumor development. To his surprise, he found the opposite: deleting PKM2 actually accelerated tumor formation and promoted liver metastasis in mice.

In his 2014 paper, Israelsen concluded that PKM2 might permit cancer cells to maintain their “plasticity,” shifting from one specialized role to another even after they’ve fully matured. In the absence of PKM2, he proposed PKM1 might take over PKM2’s influential role.

Hillis wondered if she could replicate Israelsen’s breast cancer results in a model for pancreatic cancer, especially given the conflicting findings in human data regarding PKM2 expression in the latter. Some studies suggest that high PKM2 expression correlates with accelerated disease, whiles others indicate just the opposite: that high PKM2 expression is associated with better survival rates.

“Going into the project, we were expecting similar effects in both pancreatic and breast cancer models because both cancers preferentially express PKM2, and we were using the same method of PKM2 deletion, just bred into a different cancer model,” Hillis explains. “We anticipated that PKM2 deletion would accelerate pancreatic tumor size and tumor genesis, and decrease the mouse’s lifespan. But we’ve noticed that these effects — if they exist — are very much attenuated in the pancreatic cancer model; there is only a slight decrease in lifespan and increase in tumor size without PKM2.”

Right now, her working hypothesis holds that PKM2’s influence varies depending on the tissue in question. This might explain why her own results don’t exactly parallel what Israelsen found in his breast cancer model. For instance, the method they were using to delete PKM2 is quite effective in the breast and pancreatic cells themselves, but less so in the dense scar-like tissue characteristic of pancreatic tumors in particular. It’s possible, she thinks, that this fibrous tissue may still express some PKM2 even post-knockout, perhaps hindering both a drastic decrease in lifespan and increase in tumor size.

Hillis hopes piecing together PKM2’s mechanism of action will help us better diagnose — and eventually treat — certain cancers. Her most recent results were published in the November 2017 issue Cancer & Metabolism.

Although Hillis enjoys tackling the more fundamental questions concerning cancer, she’s also interested in translating this work from bench to bedside. That’s why she decided to intern with David Ting at the Massachusetts General Hospital Cancer Center this past summer.

“I wanted to try a different type of research before applying to graduate school,” she says. “The Department of Biology frequently sends out emails about job opportunities, and there was one advertising that the Ting lab was looking for a research technician.”

Although she was still a junior at the time, she contacted Ting — an MIT alumnus with a dual degree in 7A and 10 — and together they fashioned a summer position just for her, studying the role of miniscule, fluid-filled transportation structures called exosomes in cancer development and diagnosis.

“That was the first time I’d worked with samples from actual patients,” she says. “Many of the assays were the same, but I felt closer to a clinical application than I ever had before. I really enjoy doing the foundational work to identify the basic problem, but there’s definitely something to be said for experiencing research targeted at creating a diagnostic tool. I can see the pros and cons of both approaches.”

As Hillis begins her final semester at MIT, she’s continuing her work in the Vander Heiden lab, while also finishing up the requirements for her HASS concentration in legal studies. She’s still set on pursuing a PhD in cancer biology, but the propensity to ask tough questions that drew her to science in the first place has led her to realize that the questions she raises in her own research have ramifications far beyond her lab bench. Taking policy-oriented classes in addition to her science-related ones has inspired her to pursue a law degree in conjunction with her PhD — weaving together her love for science with a newfound interest in the rules and regulations that govern how science is funded, performed, shared, applied, and monetized.

“I really enjoy doing research, and that’s something I probably will continue to do,” she says, “but I also want to influence science-related regulations, which is something I couldn’t possibly do without a law degree. I would still be heavily immersed in science, while applying the subjects I love in new and exciting ways.”

Photo credit: Raleigh McElvery
Study: Fragile X syndrome neurons can be restored

Whitehead Institute researchers are using a modified CRISPR/Cas9-guided activation strategy to investigate the most frequent cause of intellectual disability in males.

Nicole Giese Rura | Whitehead Institute
February 15, 2018

Fragile X syndrome is the most frequent cause of intellectual disability in males, affecting one out of every 3,600 boys born. The syndrome can also cause autistic traits, such as social and communication deficits, as well as attention problems and hyperactivity. Currently, there is no cure for this disorder.

Fragile X syndrome is caused by mutations in the FMR1 gene on the X chromosome, which prevent the gene’s expression. This absence of the FMR1-encoded protein during brain development has been shown to cause the overexcitability in neurons associated with the syndrome. Now, for the first time, researchers at Whitehead Institute have restored activity to the fragile X syndrome gene in affected neurons using a modified CRISPR/Cas9 system they developed that removes the methylation — the molecular tags that keep the mutant gene shut off — suggesting that this method may prove to be a useful paradigm for targeting diseases caused by abnormal methylation.

Research by the lab of Whitehead Institute for Biomedical Research Founding Member Rudolf Jaenisch, which is described online this week in the journal Cell, is the first direct evidence that removing the methylation from a specific segment within the FMR1 locus can reactivate the gene and rescue fragile X syndrome neurons.

The FMR1 gene sequence includes a series of three nucleotide (CGG) repeats, and the length of these repeats determines whether or not a person will develop fragile X syndrome: A normal version of the gene contains anywhere from 5 to 55 CGG repeats, versions with 56 to 200 repeats are considered to be at a higher risk of generating some of the syndrome’s symptoms, and those versions with more than 200 repeats will produce fragile X syndrome.

Until now, the mechanism linking the excessive repeats in FMR1 to fragile X syndrome was not well-understood. But Shawn Liu, a postdoc in Jaenisch’s lab and first author of the Cell study, and others thought that the methylation blanketing those nucleotide repeats might play an important role in shutting down the gene’s expression.

In order to test this hypothesis, Liu removed the methylation tags from the FMR1 repeats using a CRISPR/Cas9-based technique he recently developed with Hao Wu, a postdoc in the Jaenisch lab. This technique can either add or delete methylation tags from specific stretches of DNA. Removal of the tags revived the FMR1 gene’s expression to the level of the normal gene.

“These results are quite surprising — this work produced almost a full restoration of wild type expression levels of the FMR1 gene,” says Jaenisch, whose primary affiliation is with Whitehead Institute, where his laboratory is located and his research is conducted. He is also a professor of biology at MIT. “Often when scientists test therapeutic interventions, they only achieve partial restoration, so these results are substantial,” he says.

The reactivated FMR1 gene rescues neurons derived from fragile X syndrome induced pluripotent stem (iPS) cells, reversing the abnormal electrical activity associated with the syndrome. When rescued neurons were engrafted into the brains of mice, the FMR1 gene remained active in the neurons for at least three months, suggesting that the corrected methylation may be sustainable in the animal.

“We showed that this disorder is reversible at the neuron level,” says Liu. “When we removed methylation of CGG repeats in the neurons derived from fragile X syndrome iPS cells, we achieved full activation of FMR1.”

The CRISPR/Cas-9-based technique may also prove useful for other diseases caused by abnormal methylation including facioscapulohumeral muscular dystrophy and imprinting diseases.

“This work validates the approach of targeting the methylation on genes, and it will be a paradigm for scientists to follow this approach for other diseases,” says Jaenisch.

This work was supported by the National Institutes of Health, the Damon Runyon Cancer Foundation, the Rett Syndrome Research Trust, the Brain and Behavior Research Foundation, and the Helen Hay Whitney Foundation. Jaenisch is co-founder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics.

Reading and writing DNA

Department of Biology kicks off IAP seminar series with a lecture by synthetic-biology visionary George Church.

Raleigh McElvery | Department of Biology
January 31, 2018

Thanks to the invention of genome sequencing technology more than three decades ago, we can now read the genetic blueprint of virtually any organism. After the ability to read came the ability to edit — adding, subtracting, and eventually altering DNA wherever we saw fit. And yet, for George Church, a professor at Harvard Medical School, associate member of the Broad Institute, and founding core faculty and lead for synthetic biology at the Wyss Institute — who co-pioneered direct genome sequencing in 1984 — the ultimate goal is not just to read and edit, but also to write.

What if you could engineer a cell resistant to all viruses, even the ones it hadn’t yet encountered? What if you could grow your own liver in a pig to replace the faulty one you were born with? What if you could grow an entire brain in a dish? In his lecture on Jan. 24 — which opened the Department of Biology’s Independent Activities Period (IAP) seminar series, Biology at Transformative Frontiers — Church promised all this and more.

“We began by dividing the Biology IAP events into two tracks: one related to careers in academia and another equivalent track for industry,” says Jing-Ke Weng, assistant professor and IAP faculty coordinator for the department. “But then it became clear that George Church, Patrick Brown, and other speakers we hoped to invite blurred the boundaries between those two tracks. The Biology at Transformative Frontiers seminar series became about the interface of these trajectories, and how transferring technologies from lab bench to market is altering society as we know it.”

The seminar series is a staple in the Department of Biology’s IAP program, but during the past several years it has been oriented more toward quantitative biology. Weng recalls these talks as being relegated to the academic sphere, and wanted to show students that the lines between academia, industry, and scientific communication are actually quite porous.

“We chose George Church to kick off the series because he’s been in synthetic biology for a long time, and continues to have a successful academic career even while starting so many companies,” says Weng.

Church’s genomic sequencing methods inspired the Human Genome Project in 1984 and resulted in the first commercial genome sequence (the bacterium Helicobacter pylori) 10 years later. He also serves as the director of the Personal Genome Project, the “Wikipedia” of open-access human genomic data. Beyond these ventures, he’s known for his work on barcoding, DNA assembly from chips, genome editing, and stem cell engineering.

He’s also the same George Church who converted the book he co-authored with Ed Regis, “Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves,” into a four-letter code based on the four DNA nucleotides (A, T, C, and G), subsisted on nutrient broth from a lab vendor for an entire year, and dreams of eventually resurrecting woolly mammoths. He’s being featured in an upcoming Netflix Original documentary, so when he arrived at the Stata Center to give his lecture last week he was trailed by a camera crew.

According to Church, the transformative technologies that initially allowed us to read and edit DNA have grown exponentially in recent years with the invention of molecular multiplexing and CRISPR-Cas9 (think Moore’s Law but even more exaggerated). But there’s always room for improvement.

“There’s been a little obsession with CRISPR-Cas9s and other CRISPRs,” said Church. “Everybody is saying how great it is, but it’s important to say what’s wrong with it as well, because that tells us where we’re going next and how to improve on it.”

He outlined several of his own collaborations, including those aimed at devising more precise methods of genome editing, one resulting in 321 changes to the Escherichia coli genome — the largest change in any genome yet — rendering the bacterium resistant to all viruses, even those it had not yet come into contact with. The next step? Making similarly widespread changes in plants, animals, and eventually perhaps even human tissue. In fact, Church and his team have set their sights on combatting the global transplantation crisis with humanlike organs grown in animals.

“Since the dawn of transplantation as a medical practice, we’ve had to use either identical twins or rare matches that are very compatible immunologically, because we couldn’t engineer the donor or the recipient,” said Church.

Since it’s clearly unethical to engineer human donors, Church reasoned, why not engineer animals with compatible organs instead? Pigs, to be exact, since most of their organs are comparable in size and function to our own.

“This is an old dream; I didn’t originate it,” said Church. “It started about 20 years ago, and the pioneers of this field worked on it for a while, but dropped it largely because the number of changes to the genome were daunting, and there was a concern that the viruses all pigs make — retroviruses — would be released and infect the immunocompromised organ recipient.”

Church and his team successfully disrupted 62 of these retroviruses in pig cells back in 2015, and in 2017 they used these cells to generate living, healthy pigs. Today, the pigs are thriving and rearing piglets of their own. Church is also considering the prospect of growing augmented organs in pigs for human transplantation, perhaps designing pathogen-, cancer-, and age-resistant organs suitable for cryopreservation.

“Hopefully we’ll be doing nonhuman primate trials within a couple of years, and then almost immediately after that human trials,” he said.

Another possibility, rather than cultivating organs in animals for transplant, is to generate them in a dish. A subset of Church’s team is working on growing from scratch what is arguably the most complicated organ of all, the brain.

This requires differentiating multiple types of cells in the same dish so they can interact with each other to form the complex systems of communication characteristic of the human brain.

Early attempts at fashioning brain organoids often lacked capillaries to distribute oxygen and nutrients (roughly one capillary for each of the 86 billion neurons in the human brain). However, thanks to their new human transcription factor library, Church and colleagues have begun to generate the cell types necessary to create such capillaries, plus the scaffolding needed to promote the three-dimensional organization of these and additional brain structures. Church and his team have not only successfully integrated the structures with one another, but have also created an algorithm that spits out the list of molecular ingredients required to generate each cell type.

Church noted these de novo organoids are extremely useful in determining which genetic variants are responsible for certain diseases. For instance, you could sequence a patient’s genome and then create an entire organoid with the mutation in question to test whether it was the root cause of the condition.

“I’m still stunned by the breadth of projects and approaches that he’s running simultaneously,” says Emma Kowal, a second-year graduate student, member of Weng’s planning committee, and a former researcher in Church’s lab. “The seminar series is called Biology at Transformative Frontiers, and George is very much a visionary, so we thought it would be a great way to start things off.”

The four-part series also features Melissa Moore, chief scientific officer of the Moderna Therapeutics mRNA Research Platform, Jay Bradner, president of the Novartis Institutes for BioMedical Research, and Patrick Brown, CEO and founder of Impossible Foods.

How some facial malformations arise

Study explains why mutations that would seemingly affect all cells lead to face-specific birth defects.

Anne Trafton | MIT News Office
January 24, 2018

About 1 in 750 babies born in the United States has some kind of craniofacial malformation, accounting for about one-third of all birth defects.

Many of these craniofacial disorders arise from mutations of “housekeeping” genes, so called because they are required for basic functions such as building proteins or copying DNA. All cells in the body require these housekeeping genes, so scientists have long wondered why these mutations would produce defects specifically in facial tissues.

Researchers at MIT and Stanford University have now discovered how one such mutation leads to the facial malformations seen in Treacher-Collins Syndrome, a disorder that affects between 1 in 25,000 and 1 in 50,000 babies and produces underdeveloped facial bones, especially in the jaw and cheek.

The team found that embryonic cells that form the face are more sensitive to the mutation because they more readily activate a pathway that induces cell death in response to stress. This pathway is mediated by a protein called p53. The new findings mark the first time that scientists have determined how mutations in housekeeping genes can have tissue-specific effects during embryonic development.

“We were able to narrow down, at the molecular level, how issues with general regulators that are used to make ribosomes in all cells lead to defects in specific cell types,” says Eliezer Calo, an MIT assistant professor of biology and the lead author of the study.

Joanna Wysocka, a professor of chemical and systems biology at Stanford University, is the senior author of the study, which appears in the Jan. 24 online edition of Nature.

From mutation to disease

Treacher-Collins Syndrome is caused by mutations in genes that code for proteins required for the assembly and function of polymerases. These proteins, known as TCOF1, POLR1C, and POLR1D, are responsible for transcribing genes that make up cell organelles called ribosomes. Ribosomes are critical to all cells.

“The question we were trying to understand is, how is it that when all cells in the body need ribosomes to function, mutations in components that are required for making the ribosomes lead to craniofacial disorders? In these conditions, you would expect that all the cell types of the body would be equally affected, but that’s not the case,” Calo says.

During embryonic development, these mutations specifically affect a type of embryonic cells known as cranial neural crest cells, which form the face. The researchers already knew that the mutations disrupt the formation of ribosomes, but they didn’t know exactly how this happens. To investigate that process, the researchers engineered larvae of zebrafish and of an aquatic frog known as Xenopus to express proteins harboring those mutations.

Their experiments revealed that the mutations lead to impairment in the function of an enzyme called DDX21. When DDX21 dissociates from DNA, the genes that encode ribosomal proteins do not get transcribed, so ribosomes are missing key components and can’t function normally. However, this DDX21 loss only appears to happen in cells that are highly sensitive to p53 activation, including cranial neural crest cells. These cells then undergo programmed cell death, which leads to the facial malformations seen in Treacher-Collins Syndrome, Calo says.

Other embryonic cells, including other types of neural crest cells, which form nerves and other parts of the body such as connective tissue, are not affected by the loss of DDX21.

Role of DNA damage

The researchers also found that mutations of POLR1C and POLR1D also cause damage to stretches of DNA that encode some of the RNA molecules that make up ribosomes. The amount of DNA damage correlated closely with the severity of malformations seen in individual larvae, and mutations in POLR1C led to far more DNA damage than mutations in POLR1D. The researchers believe these differences in DNA damage may explain why the severity of Treacher-Collins Syndrome can vary widely among individuals.

Calo’s lab is now studying why affected cells experience greater levels of DNA damage in those particular sequences. The researchers are also looking for compounds that could potentially prevent craniofacial defects by making the cranial neural crest cells more resistant to p53-induced cell death. Such interventions could have a big impact but would have to be targeted very early in embryonic development, as the cranial neural crest cells begin forming the tissue layers that will become the face at about three weeks of development in human embryos.

The research was funded by the National Institutes of Health, Howard Hughes Medical Institute, and March of Dimes Foundation.

Biology by Numbers

Undergraduate Camilo Espinosa grew up with a love for math, before developing a second passion for immunology at MIT

Raleigh McElvery
November 10, 2017

Biology by Numbers

Person with short brown hair and green jacket stands in front of MIT pillars.

Undergraduate Camilo Espinosa grew up with a love for math, before developing a second passion for immunology at MIT

Raleigh McElvery

 

Undergraduate Camilo Espinosa, now in his senior year, tackles biological problems with the mindset of a mathematician. That’s because he initially approached the STEM fields (science, technology, engineering and mathematics) starting with the “M” and ending with the “S” — developing an appetite for math before realizing a second love for biology.

Every six months, beginning his first year of middle school, Espinosa would venture from his home on the north coast of Colombia to the nation’s capital. There, for several weeks, he would do nothing but math.

“These were math olympiads — basically a combination of math camp and competitions,” he explains. “That was the first real community I had outside my school.”

But he didn’t just glean formulas and analytical strategies from those competitions; it was his olympiad team that first introduced him to MIT. In Colombia, he explains, students must select a major almost immediately upon entering university, and are offered limited electives. MIT came to represent “academic freedom” for Espinosa, who, despite his avid and early love of math, intended to explore multiple academic avenues before limiting himself to just one.

Now, as a math and chemistry-biology double major with a concentration in philosophy, he says MIT has enabled him to pursue his many academic interests, as well as his non-academic ones. He has served as an active member of not one but four dance teams, as well as president of his fraternity. He also helped establish a channel of communication between the International Students Office and the Department of Biology, to streamline the work authorization process for international students.

He was drawn to biology, he explains, because he prefers learning processes over basic facts. “I don’t care much for memorization, but I do care about the underlying reasons for why and how things function,” he says. “I think that mindset stems from my dad.”

Espinosa’s father is an OB/GYN specializing in female oncology, who initially helped to popularize laparoscopic surgery techniques in Colombia. Often, he sees patients at a discounted price or for free if they could not afford his services.

“At the end of the day, he is just trying to help people,” Espinosa explains. “He taught me the way I think about the world. He’s the reason I do what I do, and why I’m so oriented towards the life sciences.”  

Espinosa’s siblings are studying to become doctors and veterinarians, and he himself is intrigued by the possibilities of using our body’s innate defense mechanisms to treat diseases like cancer.

His foray into the field of immunology began with antibodies — special ones taken from furry, gawky alpacas — so tiny and versatile that they can be employed for all manner of imaging, therapeutic, and diagnostic techniques. These “single domain” antibodies were a popular area of interest in Hidde Ploegh’s lab (formerly a Professor of Biology at the Whitehead Institute for Biomedical Research), where Espinosa began mid-way through October of his freshman year. Using these single-domain antibodies, the Ploegh lab had developed a treatment for melanoma in mice, and Espinosa worked to pinpoint antibodies directed against melanoma in humans.

The summer between his sophomore and junior years, Espinosa began a separate project that eventually evolved into his thesis. He honed in on one tiny antibody, known as A4, which binds to a particular protein expressed on the surface of red blood cells, and has the potential to thwart the immune response by activating a process known as “tolerance.”

Our body’s immune system is programmed to discern self from non-self, targeting foreign entities for destruction. It does so in two distinct steps. First, it creates an army of cells, that together express antibodies tailored to combat virtually every possible substance, both self and non-self. The immune system is then primed to spring into action whenever it senses something foreign, amplifying those cells that express antibodies against it. However, the body would also attack itself if not for the second step in this process: tolerance. The immune system essentially deletes the cells expressing antibodies against itself, and in doing so learns to “tolerate” its own proteins.

For example, when red blood cells die of old age (and many do every day), this triggers tolerance to the various protein components that constitute those cells — preventing related antibodies from being created.

Previous work has shown that binding a foreign protein to red blood cells triggers tolerance for that specific protein, despite being “non-self.” This could have implications for therapies to treat conditions like hemophilia, Espinosa explains, which require injections of proteins to reinstate the body’s blood-clotting abilities.

In a large proportion of patients, the immune system responds and attacks these proteins as foreign, rendering the treatment useless and barring the patients from receiving it again in the future. However, Espinosa proposes, if he could couple A4 to the treatment protein, then A4 would link the protein to the red blood cells and initiate tolerance to it. Since the body can no longer create antibodies against the treatment proteins, the therapy can run its course. In other words, the body would now see the injected proteins as self.

After months of methodical experiments, A4 didn’t appear to disguise proteins as self in the way Espinosa had initially hoped, although it did reduce the immune response triggered by the protein injection, if he staggered the protein and antibody infusions in the proper manner.

“So maybe A4 doesn’t work as a camouflage per se, but rather as a suppressor of certain immune responses,” he says. “So the results didn’t turn out exactly as we expected, but it is still a step in the right direction.”

He ultimately submitted his thesis to MIT’s Ilona Karmel Writing Prizes, earning second place in the technical writing category.  

Last summer, Espinosa explored a different side of basic research — the corporate side — during an internship at the biotechnology company Genentech. There, he investigated the pathways by which uncontrolled cell death leads to sepsis in patients.

As he wraps up his senior year and begins applying to graduate programs, Espinosa reflects on his transition from student to instructor, having served as a teaching assistant in a number of biology courses during the past three years. “As someone who arrived at MIT with a weak foundation in biology, almost everything I’ve learned since was because someone taught it to me, and taught it to me well,” he says. “I feel honored to be able to pass it on.”

Photo credit: Raleigh McElvery
Posted: 1.18.18
Combatting chemotherapy resistance

Graduate student Faye-Marie Vassel investigates a protein that helps cells tolerate DNA damage, sharing her expertise with budding scientists to further STEM education

Raleigh McElvery
December 8, 2017

Combatting chemotherapy resistance

Person with long, dark hair and lab coat stares into microscope.

Graduate student Faye-Marie Vassel investigates a protein that helps cells tolerate DNA damage, sharing her expertise with budding scientists to further STEM education

Raleigh McElvery

 

Faye-Marie Vassel has a protein. Well, as a living entity, technically she has many, but just one she affectionately refers to as her own. “My protein, REV7.” And it makes sense — if you were hard at work characterizing a single protein for all six years of your graduate career, you’d be pretty attached, too. Plus, the stakes are high. REV7, which aids in DNA damage repair, could ultimately provide insight into ways to combat chemotherapy resistance.

Although Vassel’s mother trained as an OB/GYN in Russia before moving to the U.S., serving as what Vassel describes as a “quiet” scientific role model, Vassel spent her early childhood emulating her father, a social worker, and engrossed in the social sciences. She intended to one day work in science policy — until high school when she joined an after-school program at the American Museum of Natural History in New York City, and discovered an additional interest.

Here, Vassel took a series of molecular biology classes and met her first female research mentor, a postdoctoral fellow at Rockefeller University, who encouraged her to participate in another, more advanced science program funded by the National Science Foundation.

“I initially had my doubts, but just having that support changed everything,” Vassel says. “That was my first time doing research of any kind, and I got a sense of the sheer diversity of potential research projects. That’s also when I heard there was something called biophysics.”

From that point on, Vassel was hooked. As an undergraduate at Stony Brook University, she initially declared a major in physics before switching to biochemistry. Later, when it came time to select a graduate school, she was split between MIT and the University of California, Berkeley. As she recalls, MIT’s graduate preview weekend made all the difference.

“I had the chance to stay with biology students and speak with professors,” she says. “The whole experience made the department seem personal, and demystified the graduate school process by making it more tangible.”

She proposed a joint position between two labs: Graham Walker’s lab, based in Building 68, and Michael Hemann’s lab situated in the Koch Institute for Integrative Cancer Research. Walker’s lab focuses on microbiology, DNA repair, and antibiotic resistance, while Hemann’s lab investigates chemotherapy resistance in hopes of improving cancer therapies. After stumbling upon one of their joint papers, Vassel decided she’d like to combine the two.

“It’s invaluable to have both perspectives,” she says. “Mike’s lab just celebrated its 10th anniversary, while Graham‘s just had its 35th. It’s been interesting seeing the different ways they approach their respective research questions, because they were trained in such different scientific eras.”

Although Vassel is currently the only student formally working in both labs, the collaboration between Walker and Hemann, aimed at combatting chemotherapy resistance, has been ongoing.

Frontline chemotherapies, including one anticancer agent called cisplatin, kill cancer cells by damaging their DNA and preventing them from synthesizing new genetic material. Just how sensitive cancer cells are to cisplatin — and therefore how effective the treatment is — depends on whether the cell can repair the damage and bypass DNA-damage induced cell death. In some cases, cells increase production of “translesion polymerases,” which are specialized DNA polymerases that can help cells tolerate certain kinds of DNA damage by synthesizing across from damaged DNA or DNA bound to a carcinogen.

Vassel’s protein, REV7, is a structural subunit of one key translesion polymerase, and its expression is deregulated in many different cancer cells. As Vassel suggests, if one aspect of these translesion polymerases — say, the REV7 subunit — could be altered to hinder repair, then perhaps cancer-ridden cells could regain drug sensitivity.

Thanks to recently-developed CRISPR-Cas9 gene editing techniques, Vassel has removed REV7 entirely from drug resistant lung cancer cells, and watched as cisplatin sensitivity was restored. She also conducted rescue experiments, adding REV7 back into cell lines lacking the protein to see whether those cells become resistant to the drug once again. Most recently, she has been working in murine models to see whether REV7 has similar effects in a living system.

If her hypothesis is correct, REV7 would be a powerful target for drug development. Treatments that inhibit REV7, she explains, could be used in tandem with frontline chemotherapies like cisplatin to prevent resistance.

Since her foray into biology at the American Museum of Natural History almost a decade ago, Vassel has maintained her passion for science outreach. During her time at MIT, she has served as a math tutor for middle schoolers in the Cambridge public school system. She also volunteered as a science and math mentor for high school students, as part of a dual athletic and academic program founded by MIT.

As Vassel wraps up her final year of graduate studies, she is torn between completing an academic postdoc and indulging her early interest in science education policy.

“Growing up in New York City, it was not lost on me that — despite the city’s wonderful diversity — people from historically underserved groups were still missing from many science-related positions,” Vassel says. “It got me thinking about the dire need for policymakers to improve curricula to make science more inclusive of all life experiences. There’s this idea that science is apolitical when it’s really not, and that mindset can have detrimental effects on equity and diversity in science.”

Photo credit: Raleigh McElvery