Research Area: Human Disease

Parasite’s riff on essential enzyme highlights unique biology
Nicole Giese Rura | Whitehead Institute
September 18, 2018

Cambridge, Mass. — The primary currency of energy in cells—adenosine triphosphate (ATP)—is essential for their survival and without it, cellular processes would seize. In the apicomplexan Toxoplasma gondii (T. gondii), a parasite that Whitehead Member Sebastian Lourido studies, key components of the ATP synthase—the enzyme responsible for ATP production—have remained elusive. While investigating indispensable proteins with unknown functions, Lourido and Diego Huet, a postdoctoral researcher in Lourido’s lab, identified a critical component of the enzyme. While highly conserved from yeast to humans, it proved to be considerably different in T. gondii. The findings, published online September 11 in the journal eLife, underscore the unique biology of these parasites and highlight differences between them and their human hosts.

More closely related to plants than to animals, the single-celled apicomplexans are among the most common and deadly human pathogens. According to the World Health Organization, every year these diseases sicken hundreds of millions, kill hundreds of thousands—primarily children—and cost billions of dollars to treat. Species of apicomplexans cause malaria (Plasmodium spp.), cryptosporidiosis (Cryptosporidium spp.), and toxoplasmosis (T. gondii).

Using a CRISPR-based genetic screen that they had adapted to T. gondii, Lourido and Huet had previously identified about 200 genes in T. gondii that are fitness-conferring and specific to apicomplexans. Of that cadre, a few were localized to the mitochondria, where cells manufacture ATP, the cellular currency of energy. Because those genes have not been annotated previously, and the proteins encoded by them have no known function, Huet ran their protein sequences through a database that compared them to protein sequences with known structures.

One of the proteins came back with an interesting hit: it shares structural similarity, but not sequence similarity, with an integral part of the ATP synthase. Most of the protein subunits that compose the apicomplexan ATP synthase have been identified, but key components of the stator—a portion of the enzyme essential for its function—was not yet known.

When Huet experimentally removed the function of the stator subunit in T. gondii, the parasites’ growth stalled, their mitochondria were misshapen and shrunken, and energy production halted—all traits typical of interrupted ATP synthase function.

Because the apicomplexan ATP synthase varies so much from its hosts’ version, those differences, like the unusual stator, could serve as future drug targets. But for Lourido, who is also an assistant professor of biology at Massachusetts Institute of Technology (MIT), the unique stator protein emphasizes how unique and extraordinary apicomplexan organisms are compared to us and their other hosts.

This work was supported by the National Institutes of Health (NIH grants 1DP5OD017892, R21AI123746, and K99AI137218).

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Sebastian Lourido’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also an assistant professor of biology at Massachusetts Institute of Technology.

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Full Citation:

“Identification of cryptic subunits from an apicomplexan ATP synthase”

eLife, online September 11, 2018.  DOI: 10.7554/eLife.38097

Diego Huet (1) , Esther Rajendran (2) , Giel G van Dooren (2) , Sebastian Lourido (1,3*).

1. Whitehead Institute for Biomedical Research, Cambridge, United States

2. Research School of Biology, Australian National University, Canberra, Australia

3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

Ankur Jain

Education

  • PhD, 2013, University of Illinois, Urbana-Champaign
  • BTech, 2007,  Biotechnology and Biochemical Engineering, Indian Institute of Technology Kharagpur

Research Summary

We study how biomolecules in a cell self-organize. In particular, we are interested in understanding how membrane-free cellular compartments such as RNA granules form and function. Our lab develops new biochemical and biophysical techniques to investigate these compartments and to understand their dysfunction in human disease.

Awards

  • Young Alumni Achiever’s Award, Indian Institute of Technology Kharagpur, 2019
  • NIH K99/R00 Pathway to Independence Award, 2017
  • Pew Scholar in the Biomedical Sciences, 2022
Study suggests glaucoma may be an autoimmune disease

Unexpected findings show that the body’s own immune system destroys retinal cells.

Anne Trafton | MIT News Office
August 11, 2018

Glaucoma, a disease that afflicts nearly 70 million people worldwide, is something of a mystery despite its prevalence. Little is known about the origins of the disease, which damages the retina and optic nerve and can lead to blindness.

A new study from MIT and Massachusetts Eye and Ear has found that glaucoma may in fact be an autoimmune disorder. In a study of mice, the researchers showed that the body’s own T cells are responsible for the progressive retinal degeneration seen in glaucoma. Furthermore, these T cells appear to be primed to attack retinal neurons as the result of previous interactions with bacteria that normally live in our body.

The discovery suggests that it could be possible to develop new treatments for glaucoma by blocking this autoimmune activity, the researchers say.

“This opens a new approach to prevent and treat glaucoma,” says Jianzhu Chen, an MIT professor of biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and one of the senior authors of the study, which appears in Nature Communications on Aug. 10.

Dong Feng Chen, an associate professor of ophthalmology at Harvard Medical School and the Schepens Eye Research Institute of Massachusetts Eye and Ear, is also a senior author of the study. The paper’s lead authors are Massachusetts Eye and Ear researchers Huihui Chen, Kin-Sang Cho, and T.H. Khanh Vu.

Genesis of glaucoma

One of the biggest risk factors for glaucoma is elevated pressure in the eye, which often occurs as people age and the ducts that allow fluid to drain from the eye become blocked. The disease often goes undetected at first; patients may not realize they have the disease until half of their retinal ganglion cells have been lost.

Most treatments focus on lowering pressure in the eye (also known as intraocular pressure). However, in many patients, the disease worsens even after intraocular pressure returns to normal. In studies in mice, Dong Feng Chen found the same effect.

“That led us to the thought that this pressure change must be triggering something progressive, and the first thing that came to mind is that it has to be an immune response,” she says.

To test that hypothesis, the researchers looked for immune cells in the retinas of these mice and found that indeed, T cells were there. This is unusual because T cells are normally blocked from entering the retina, by a tight layer of cells called the blood-retina barrier, to suppress inflammation of the eye. The researchers found that when intraocular pressure goes up, T cells are somehow able to get through this barrier and into the retina.

The Mass Eye and Ear team then enlisted Jianzhu Chen, an immunologist, to further investigate what role these T cells might be playing in glaucoma. The researchers generated high intraocular pressure in mice that lack T cells and found that while this pressure induced only a small amount of damage to the retina, the disease did not progress any further after eye pressure returned to normal.

Further studies revealed that the glaucoma-linked T cells target proteins called heat shock proteins, which help cells respond to stress or injury. Normally, T cells should not target proteins produced by the host, but the researchers suspected that these T cells had been previously exposed to bacterial heat shock proteins. Because heat shock proteins from different species are very similar, the resulting T cells can cross-react with mouse and human heat shock proteins.

To test this hypothesis, the team brought in James Fox, a professor in MIT’s Department of Biological Engineering and Division of Comparative Medicine, whose team maintains mice with no bacteria. The researchers found that when they tried to induce glaucoma in these germ-free mice, the mice did not develop the disease.

Human connection

The researchers then turned to human patients with glaucoma and found that these patients had five times the normal level of T cells specific to heat shock proteins, suggesting that the same phenomenon may also contribute to the disease in humans. The researchers’ studies thus far suggest that the effect is not specific to a particular strain of bacteria; rather, exposure to a combination of bacteria can generate T cells that target heat shock proteins.

One question the researchers plan to study further is whether other components of the immune system may be involved in the autoimmune process that gives rise to glaucoma. They are also investigating the possibility that this phenomenon may underlie other neurodegenerative disorders, and looking for ways to treat such disorders by blocking the autoimmune response.

“What we learn from the eye can be applied to the brain diseases, and may eventually help develop new methods of treatment and diagnosis,” Dong Feng Chen says.

The research was funded by the National Institutes of Health, the Lion’s Foundation, the Miriam and Sheldon Adelson Medical Research Foundation, the National Nature Science Foundation of China, the Ivan R. Cottrell Professorship and Research Fund, the Koch Institute Support (core) Grant from the National Cancer Institute, and the National Eye Institute Core Grant for Vision Research.

Researchers discover new type of lung cell, critical insights for cystic fibrosis

A comprehensive single-cell analysis of airway cells in mice, validated in human tissue, reveals molecular details critical to understanding lung disease.

Karen Zusi
August 1, 2018

Researchers have identified a rare cell type in airway tissue, previously uncharacterized in the scientific literature, that appears to play a key role in the biology of cystic fibrosis. Using new technologies that enable scientists to study gene expression in thousands of individual cells, the team comprehensively analyzed the airway in mice and validated the results in human tissue.

Led by researchers from the Broad Institute of MIT and Harvard and Massachusetts General Hospital (MGH), the molecular survey also characterized gene expression patterns for other new cell subtypes. The work expands scientific and clinical understanding of lung biology, with broad implications for all diseases of the airway — including asthma, chronic obstructive pulmonary disease, and bronchitis.

Jayaraj Rajagopal, a physician in the Pulmonary and Critical Care Unit at MGH, associate member at the Broad Institute, and a Howard Hughes Medical Institute (HHMI) faculty scholar, and Broad core institute member Aviv Regev, director of the Klarman Cell Observatory at the Broad Institute, professor of biology at MIT, and an HHMI investigator, supervised the research. Daniel Montoro, a graduate student in Rajagopal’s lab, and postdoctoral fellows Adam Haber and Moshe Biton in the Regev lab are co-first authors on the paper published today in Nature.

“We have the framework now for a new cellular narrative of lung disease,” said Rajagopal, who is also a professor at Harvard Medical School and a principal faculty member at the Harvard Stem Cell Institute. “We’ve uncovered a whole distribution of cell types that seem to be functionally relevant. What’s more, genes associated with complex lung diseases can now be linked to specific cells that we’ve characterized. The data are starting to change the way we think about lung diseases like cystic fibrosis and asthma.”

“With single-cell sequencing technology, and dedicated efforts to map cell types in different tissues, we’re making new discoveries — new cells that we didn’t know existed, cell subtypes that are rare or haven’t been noticed before, even in systems that have been studied for decades,” said Regev, who is also co-chair of the international Human Cell Atlas consortium. “And for some of these, understanding and characterizing them sheds new light immediately on what’s happening inside the tissue.”

Using single-cell RNA sequencing, the researchers analyzed tens of thousands of cells from the mouse airway, mapping the physical locations of cell types and creating a cellular “atlas” of the tissue. They also developed a new method called pulse-seq to monitor development of cell types from their progenitors in the mouse airway. The findings were validated in human tissue.

One extremely rare cell type, making up roughly one percent of the cell population in mice and humans, appeared radically different from other known cells in the dataset. The team dubbed this cell the “pulmonary ionocyte” because its gene expression pattern was similar to ionocytes — specialized cells that regulate ion transport and hydration in fish gills and frog skin.

Strikingly, at levels higher than any other cell type, these ionocytes expressed the gene CFTR — which, when mutated, causes cystic fibrosis in humans. CFTR is critical for airway function, and for decades researchers and clinicians have assumed that it is frequently expressed at low levels in ciliated cells, a common cell type spread throughout the entire airway.

But according to the new data, the majority of CFTR expression occurs in only a few cells, which researchers didn’t even know existed until now.

When the researchers disrupted a critical molecular process in pulmonary ionocytes in mice, they observed the onset of key features associated with cystic fibrosis — most notably, the formation of dense mucus. This finding underscores how important these cells are to airway-surface regulation.

“Cystic fibrosis is an amazingly well-studied disease, and we’re still discovering completely new biology that may alter the way we approach it,” said Rajagopal. “At first, we couldn’t believe that the majority of CFTR expression was located in these rare cells, but the graduate students and postdocs on this project really brought us along with their data.”

The results may also have implications for developing targeted cystic fibrosis therapies, according to the team. For example, a gene therapy that corrects for a mutation in CFTRwould need to be delivered to the right cells, and a cell atlas of the tissue could provide a reference map to guide that process.

The study further highlighted where other disease-associated genes are expressed in the airway. For example, asthma development has been previously linked with a gene that encodes a sensor for rhinoviruses, and the data now indicate that this gene is expressed by ciliated cells. Another gene linked with asthma is expressed in tuft cells, which separated into at least two groups — one that senses chemicals in the airway and one that produces inflammation. The results suggest that a whole ensemble of cells may be responsible for different aspects of asthma.

Using the pulse-seq assay, the researchers tracked how the newly characterized cells and subtypes in the mouse airway develop. They demonstrated that mature cells in the airway arise from a common progenitor: the basal cells. The team also discovered a previously undescribed cellular structure in the tissue. These structures, which the researchers called “hillocks,” are unique zones of rapid cell turnover, and their function is not yet understood.

“The atlas that we’ve created is already starting to drastically re-shape our understanding of airway and lung biology,” said Regev. “And, for this and other organ systems being studied at the single-cell level, we’ll have to drape everything we know on top of this new cellular diversity to understand human health and disease.”

Funding for this study was provided in part by the Klarman Cell Observatory at the Broad Institute, Manton Foundation, HHMI, New York Stem Cell Foundation, Harvard Stem Cell Institute, Human Frontiers Science Program, and National Institutes of Health.

Paper(s) cited:

Montoro DT, Haber AL, Biton M et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature. Online August 1, 2018. DOI: 10.1038/s41586-018-0393-7

Sharpening the edges of cancer chemotherapy
Nicole Davis | Whitehead Institute
July 11, 2018

Cambridge, MA — Tackling unsolved problems is a cornerstone of scientific research, propelled by the power and promise of new technologies. Indeed, one of the shiniest tools in the biomedical toolkit these days is the genome editing system known as CRISPR/Cas9. Whitehead Institute Member David Sabatini and his colleagues pioneered the use of this tool as a foundation for large-scale genetic screens in human cells, turning up a treasure trove of new insights into cellular metabolism, in both normal cells and cancer cells.

When Naama Kanarek, a postdoc in Sabatini’s laboratory, pondered how to apply these state-of-the-art CRISPR/Cas9 screens to her own research, her thoughts turned to a classic cancer chemotherapy drug, methotrexate, which has been in clinical use for nearly seven decades. Often used to treat a form of pediatric leukemia, known as acute lymphoblastic leukemia (ALL), the drug, when deployed as part of a multifaceted treatment plan, can be highly effective. But its power comes at a cost. Because methotrexate can damage not only cancer cells but also healthy tissues, it must be administered with great care. For children who receive high doses of the drug, a mainstay of ALL treatment, that can mean several days spent in the hospital with rigorous clinical monitoring.

In other forms of cancer, methotrexate’s efficacy is more uncertain. For example, in pediatric osteosarcoma, only 65 percent of patients respond. Unfortunately, there is currently no way for doctors to pinpoint who will and who will not.

“From a scientific standpoint, methotrexate is quite special because it was the first metabolic drug to be developed, but much of its biology remains to be discovered — particularly what drives these different responses in patients,” Kanarek says. “So, this is really one of these old, classic questions that has been lingering in the field for some time. We thought we could learn something new.”

And they did. In the July 11 online issue of the journal Nature, Kanarek, Sabatini, and their colleagues report the findings of a CRISPR/Cas9 screen for factors involved in methotrexate sensitivity. The team’s work yielded a surprising set of discoveries that point to the breakdown of histidine — one of several amino acids used by the body to construct proteins — as a critical gatekeeper of cancer cells’ vulnerability to methotrexate. The researchers’ findings not only help illuminate the biology of a well-known cancer chemotherapy, but also suggest a simple dietary supplement that could help broaden its therapeutic window and reduce its toxicity.

 “This study is an example of the power of modern genomic tools to shine a bright light on longstanding questions in human biology,” says senior author David Sabatini, a Member of Whitehead Institute, a professor of biology at Massachusetts Institute of Technology and investigator with the Howard Hughes Medical Institute (HHMI). “While cancer chemotherapies can be quite effective, their biological effects are often poorly understood. By laying bare their biology, we may be able to devise ways to utilize them more wisely.”

ATTACK THE CANCER, NOT THE PATIENT

The history of methotrexate stretches back to the 1940s, a time when strikingly little was known about the origins of cancer much less how best to treat it. The birth of methotrexate as a chemotherapeutic agent was sparked by the astute observations of Sidney Farber, a pediatric pathologist at Boston Children’s Hospital who cared for children with a variety of maladies, including ALL. In the course of caring for patients with ALL, Farber recognized that cancer cells depended on the nutrient folic acid for their own proliferation. That gave him the idea of using folate antagonists to treat ALL. Methotrexate was developed in 1949 precisely for this purpose and was subsequently shown to induce remission in children with ALL. Fast forward to today, and the drug has evolved into a significant tool in oncologists’ toolkit.

“Methotrexate is a major part of the backbone of chemotherapy treatment across many human cancers,” says Loren Walensky, a pediatric hematologist/oncologist at the Dana-Farber Cancer Institute who is not a study co-author but served as an early adviser on the project and will also play a deeper role in planning future follow-up studies. “It is also used outside of the cancer field for the treatment of several autoimmune diseases.”

He added, “But as with all chemotherapy, the critical issue is how to best use it to inflict maximal damage on the cancer without irreparably harming the patient.”

Kanarek explains how new genetic tools are allowing insights into the sensitivity of cancer cells to methotrexate.

The basic mechanics of methotrexate are fairly well known. The drug inhibits dihydrofolate reductase (DHFR), an enzyme that generates the functional form of folate, known as tetrahydrofolate (THF). THF is essential for preparing the raw materials needed to make nucleic acids, such as DNA, which carries cells’ genetic information, and RNA, a close chemical relative involved in making proteins. “Proliferating cells must duplicate their DNA, so they need a lot of THF,” Kanarek explains. “But even cells that are not dividing need to make RNA, and that requires THF, too.”

The results of Kanarek’s CRISPR/Cas9 screen now bring greater clarity to this molecular picture. She and her colleagues uncovered another enzyme, called FTCD, which is involved in the breakdown of histidine. Interestingly, FTCD also requires THF for its function — though not nearly as much as the main target of methotrexate, DHFR. Despite the differential demands of the two enzymes, they both draw from the same, shared pool of THF.

“Under normal conditions, this pool is sufficiently full, so there is no competition for resources, even in rapidly dividing cells,” Kanarek says.

But when the amount of THF becomes limiting — as it does in cells that are treated with methotrexate — the story is quite different, the Whitehead Institute team discovered. In that case, the activity of FTCD poses serious problems, because there isn’t enough THF in the pool to support both cell proliferation and histidine breakdown. When that happens, the cells die.

That got Kanarek thinking more about histidine: Could the nutrient provide a way to tinker with FTCD activity and, by virtue of the cancer cells’ own metabolism, make them more vulnerable to methotrexate?

To explore this question, the researchers used mouse models of leukemia, engineered by transplanting human leukemia cells under the skin of immunocompromised mice. A subset of the mice received injections of methotrexate together with histidine. This one-two punch, Kanarek hypothesized, should ramp up the function of FTCD and more rapidly drain the THF pool, thereby making the cells more sensitive to the cancer-killing effects of methotrexate.

That is precisely what the team observed. Notably, these experiments involved lower than normal doses of methotrexate, suggesting the cells had indeed been made more sensitive to the cancer drug. Moreover, the studies included a human leukemia cell line, called SEM, which harbors a specific genetic mutation that is associated with a particularly poor prognosis in patients — further underscoring the power of the histidine degradation pathway to weaken cells’ defenses.

Now, Kanarek and her colleagues are working to extend these initial findings with additional preclinical studies and, together with Walensky, determine how to best evaluate the potential benefits of histidine supplementation in cancer patients. Their ultimate goal: to pursue clinical trials that will assess histidine’s ability to improve the effectiveness of methotrexate in humans.

In addition to making cancer cells more vulnerable to methotrexate, the Whitehead Institute team’s research also holds promise for another therapeutic challenge: identifying which patients will or will not respond to the drug.

Two other enzymes cooperate with FTCD in breaking down histidine. The levels of one of the enzymes, known as HAL, appears to correlate with cells’ sensitivity to methotrexate: That is, cancer cells with high levels of HAL tend to be more sensitive to the drug. More work is needed to determine whether this correlation extends to a broader swath of patient samples and if it has predictive value in the clinic. Nevertheless, Kanarek and her colleagues are already beginning work on this front. Together with Abner Louissaint, Jr., a hematopathologist at Massachusetts General Hospital who also served as an early adviser on the Nature study, the Whitehead Institute team will launch a second clinical study to examine whether HAL levels can predict methotrexate response in patients with lymphoma.

“Being able to understand who is going to respond to methotrexate and who is not, and how to achieve a therapeutic benefit while mitigating the drug’s potential side effects, could have a profound impact on patient care,” Walensky says. “The insights from this study bring an entirely new dimension to our understanding of a decades-old and critically important cancer medicine. And as a physician and a scientist, that’s truly exciting.”

Written by Nicole Davis

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David Sabatini’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a Howard Hughes Medical Institute investigator and a professor of biology at Massachusetts Institute of Technology.

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Full citation:

“Histidine catabolism is a major determinant of methotrexate sensitivity”

Nature, online on July 11, 2018.

Naama Kanarek (1,2,3,4), Heather R. Keys (1), Jason R. Cantor (1,2,3,4), Caroline A. Lewis (1), Sze Ham Chan (1), Tenzin Kunchok (1), Monther Abu-Remaileh (1,2,3,4), Elizaveta Freinkman (1), Lawrence D. Schweitzer (4), and David M. Sabatini (1,2,3,4).

  1. Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Department of Biology, 455 main Street, Cambridge, Massachusetts 02142, USA
  2. Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
  3. Koch Institute for Integrative Cancer Research and Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA
  4. Broad Institute of Harvard and Massachusetts Institute of Technology, 415 main Street, Cambridge, Massachusetts 02142, USA
Restricting a key cellular nutrient could slow tumor growth

Researchers identify the amino acid aspartate as a metabolic limitation in certain cancers.

Raleigh McElvery | Department of Biology
June 29, 2018

Remove tumor cells from a living organism and place them in a dish, and they will multiply even faster than before. The mystery of why this is has long stumped cancer researchers, though many have simply focused on the mutations and chains of molecular reactions that could prompt such a disparity. Now, a group of MIT researchers suggests that the growth limitations in live organisms may stem from a different source: the cell’s environment. More specifically, they found that the amino acid aspartate serves as a key nutrient needed for the “proliferation” or rapid duplication of cancer cells when oxygen is not freely available.

The biologists took cancer cells from various tissue types and engineered them to convert another, more abundant substrate into aspartate using the gene encoding an enzyme from guinea pigs. This had no effect on the cells sitting in a dish, but the same cells implanted into mice engendered tumors that grew faster than ever before. The researchers had increased the cells’ aspartate supply, and in doing so successfully sped up proliferation in a living entity.

“There hasn’t been a lot of thought into what slows tumor growth in terms of the cellular environment, including the sort of food cancer cells need,” says Matthew Vander Heiden, associate professor of biology, associate director of the Koch Institute for Integrative Cancer Research, and senior author of the study. “For instance, if you’re trying to get to a given destination and I want to slow you down, my best bet is to set up a roadblock at a place on your route where you’d experience a slow-down anyways, like a long traffic light. That’s essentially what we’re interested in here — understanding what nutrients the cell is already lacking that put the brakes on proliferation, and then further limiting those nutrients to inhibit growth even more.”

Lucas Sullivan, a postdoc in Vander Heiden’s lab, is the lead author of the study, which appeared in Nature Cell Biology on June 25.

Building the case for aspartate

Isolating a single factor that could impact tumor growth within an organism is tricky business. One potential candidate came to Sullivan via a paper he co-authored with graduate student Dan Gui in 2015, which asked a somewhat controversial question: Why is it that cells need to consume oxygen through cellular respiration in order to proliferate?

It’s a rather counter-intuitive question, because some scientific literature suggests just the opposite: Cancer cells in an organism (“in vivo”) do not enjoy the same access to oxygen as they would in a dish, and therefore don’t depend on oxygen to produce enough energy to divide. Instead, they switch to a different process, fermentation, that doesn’t require oxygen. But Sullivan and Gui noted that cancer cells do rely on oxygen for another reason: to produce aspartate as a byproduct.

Aspartate, they soon confirmed, does, in fact, play a crucial role in controlling the rate of cancer cell proliferation. In another study one year later, Sullivan and Gui noted that the antidiabetic drug metformin, known to inhibit mitochondria, slowed tumor growth and decreased aspartate levels in cells in vivo. Since mitochondria are key to cellular respiration, Sullivan reasoned that blocking their function in an already oxygen-constrained environment (the tumor) might make cancer cells vulnerable to further suppression of respiration — and aspartate — explaining why metformin seems to have such a strong effect on tumor growth.

Despite being potentially required for certain amino acids and the synthesis of all four DNA nucleotides, aspartate is already hard to come by, even in oxygen-rich environments. It’s among the lowest concentration amino acids in our blood, and has no way to enter our cells unless a rare protein transporter is present. Precisely why aspartate import is so inefficient remains an evolutionary mystery; one possibility is that its scarcity serves as a “failsafe,” preventing cells from multiplying until they have all the resources to properly do so.

Regardless, the easiest way for cells to get aspartate is not to import it from outside, but rather to make it directly inside, breaking down another amino acid called asparagine to generate it. However, there are very few known mammals that have an enzyme capable of producing aspartate from asparagine — among them, the guinea pig.

Channeling the guinea pig

In the 1950s, a researcher named John Kidd made an accidental discovery. He injected cancer-ridden rats with sera from various animals — rabbits, horses, guinea pigs, and the like — and discovered that guinea pig serum alone shrunk the rats’ tumors. It wasn’t until years later that scientists learned it was an enzyme in the guinea pig blood called guinea pig asparaginase 1 (gpASNase1) that was responsible for this antitumorigenic effect. Today, we know about a host of simpler organisms with similar enzymes, including bacteria and zebrafish. In fact, bacterial asparaginase is approved as a medicine to treat acute lymphocytic leukemia.

Because guinea pigs are mammals and thus have similar metabolisms to our own, the MIT researchers decided to use gpASNase1 to increase aspartate levels in tumors in four different tumor types and ask whether the tumors would grow faster. This was the case for three of the four types: The colon cancer cells, osteosarcoma cells, and mouse pancreatic cancer cells divided more rapidly than before, but the human pancreatic cancer cells continued to proliferate at their normal pace.

“This is a relatively small sample, but you could take this to mean that not every cell in the body is as sensitive to loss of aspartate production as others,” Sullivan says. “Acquiring aspartate may be a metabolic limitation for only a subset of cancers, since aspartate can be produced via a number of different pathways, not just through asparagine conversion.”

When the researchers tried to slow tumor growth using the antidiabetic metformin, the cells expressing gpASNase1 remained unaffected — confirming Sullivan’s prior suspicion that metformin slows tumor growth specifically by impeding cellular respiration and suppressing aspartate production.

“Our initial finding connecting metformin and proliferation was very serendipitous,” he says, “but these most recent results are a clear proof of concept. They show that decreasing aspartate levels also decreases tumor growth, at least in some tumors. The next step is to determine if there are other ways to more intentionally target aspartate synthesis in certain tissues and improve our current therapeutic approaches.”

Although the efficacy of using metformin to treat cancer remains controversial, these findings indicate that one means to target tumors would be to prevent them from accessing or producing nutrients like aspartate to make new cells.

“Although there are many limitations to cancer cell proliferation, which metabolites become limiting for tumor growth has been poorly understood,” says Kivanc Birsoy, the Chapman-Perelman Assistant Professor at Rockefeller University. “This study identifies aspartate as one such limiting metabolite, and suggests that its availability could be targeted for anti-cancer therapies.”

Birsoy is a former postdoc in professor of biology David Sabatini’s lab, who authored a paper published in the same issue of Nature Cell Biology, identifying aspartate as a major growth limitation in oxygen-deprived tumors.

“These companion papers demonstrate that some tumors in vivo are really limited by the chemical processes that require oxygen to get the aspartate they need to grow, which can affect their sensitivity to drugs like metformin,” Vander Heiden says. “We’re beginning to realize that understanding which cancer patients will respond to which treatments may be determined by factors besides genetic mutations. To really get the full picture, we need to take into account where the tumor is located, its nutrient availability, and the environment in which it lives.”

The research was funded by an NIH Pathway to Independence Award, the American Cancer Society, Ludwig Center for Molecular Oncology Fund, the National Science Foundation, a National Institutes of Health Ruth Kirschstein Fellowship, Alex’s Lemonade Stand Undergraduate Research Fellowship, Damon Runyon Cancer Research Foundation, Howard Hughes Medical Institute Faculty Scholar Award, Stand Up to Cancer, Lustgarten Foundation, Ludwig Center at MIT, the National Institutes of Health, and the Koch Institute’s Center for Precision Cancer Medicine.

Stem cell-derived zika model suggests mechanisms underlying microcephaly
Nicole Giese Rura | Whitehead Institute
June 21, 2018

Cambridge, MA  – Scientists turn to model organisms, like mice and yeast, to investigate the biology underlying emerging diseases. But for the Zika virus, the lack of a good model hampered this type of research. Now, a team of researchers in the laboratory of Whitehead Institute Founding Member Rudolf Jaenisch has devised a way to model Zika and other neural diseases in a dish. Their work is described this week in the journal PNAS.

The Zika virus was identified in 1947 in Uganda, but a 2013 epidemic in French Guinea first brought it to the public’s attention. As the disease spread throughout the Americas and the Caribbean in 2014, abnormalities, such as microcephaly in newborns, were increasingly reported when mothers were infected during their first trimester. Scientists’ efforts to better understand the virus and its mechanisms quickly hit a snag: mice, which are often used to model disease pathology, are not vulnerable to the Zika virus unless their innate immune defenses are knocked out. Additionally, neural diseases, such as those that cause microcephaly, affect cells that reside deep in the brain, and they cannot be easily accessed for observation and manipulation.

In order to circumvent these challenges and to model Zika in the lab, the researchers turned to induced pluripotent stem cells (iPSCs)–adult cells that have been pushed back to a embryonic stem cell-like state. iPSCs can in turn be nudged to mature into almost any cell type in the body. In previous work, Julien Muffat and Yun Li, former postdoctoral researchers in the Jaenisch lab, were the first to use iPSCs to create microglia, the specialized immune cells that maintain the brain and spinal cord and care for them after injury.

In the current work, Muffat and Li teamed up with Attya Omer, also a graduate student in the Jaenisch lab, and Lee Gehrke’s lab at MIT to study the effect of the Zika virus on iPSC-derived versions of three neural cell types critical during human fetal brain development: microglia, neural progenitors, and astrocytes. Whether the Zika virus can infect these cells and how well the cells can clear the virus could provide insight into why the virus can cause birth defects like microcephaly. Using their model, the team determined that after being infected with a strain derived from the initial Ugandan Zika virus, microglia can survive and can continue to harbor the virus. This is important because in a developing embryo, microglia move from the yolk sac to the developing brain very early in gestation. The study shows that, like their in vivo counterparts, iPSC-derived microglia could invade the immature neural tissue of a brain organoid, and pre-infected microglia could transfer the virus to the organoids. According to Muffat, this suggests that if microglial precursors are infected before their journey, they could shuttle the Zika virus to the developing brain and infect the neural progenitors residing there.

Neural progenitor cells, which during gestation produce the neurons and glia that constitute the majority of the human brain, are particularly vulnerable to the Zika virus and die when infected. To better understand why these cells are so susceptible, the team compared how the Zika virus and the closely related dengue virus affect the neural progenitor cells. Dengue, which does not cause birth defects like microcephaly, triggers a strong cellular immune response, called interferon, in the neural progenitors, which enables the progenitor cells to efficiently fight and clear the dengue virus. In sharp contrast, when exposed to the Zika virus, neural progenitors mount little if any interferon immune defense. Pretreating the neural progenitor cells with interferon before exposure to the Zika virus impedes the virus’s progression and proliferation, and reduces cell death. These results suggest that therapeutically altering interferon levels could prevent some of the more dire effects of Zika infection on the neural progenitor cells.

According to the team, using iPSC-derived cells has great potential for modeling Zika virus as well as many other diseases that affect the central nervous system.

This work was supported by the European Leukodystrophy Association, the Brain & Behavior Research Foundation, the Simons Foundation (SFARI 204106), the International Rett Syndrome Foundation, Howard Hughes Medical Institute, the National Institutes of Health (NIH grants HD 045022, R37-CA084198, AI100190), the ELA Foundation, the Emerald Foundation, and Biogen. Jaenisch is a cofounder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics.

Written by Nicole Giese Rura
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Rudolf Jaenisch’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.
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Full citation:
“Human Induced Pluripotent Stem Cell-derived Glial Cells and Neural Progenitors Display Divergent Responses to Zika and Dengue Infections”
PNAS, online June 18, 2018.
Julien Muffat (1,8), Yun Li (1,8), Attya Omer (1,8), Ann Durbin (3,4,5), Irene Bosch (3,4,5), Grisilda Bakiasi (6), Edward Richards (7), Aaron Meyer (7), Lee Gehrke (3,4,5), Rudolf Jaenisch (1,2).
1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, Cambridge, MA 02139, USA
3. IMES, Massachusetts Institute of Technology, Cambridge MA 02139, USA
4. Department of Microbiology and Immunobiology, Harvard Medical School, Boston 02115, USA
5. Harvard-MIT Program in Health Sciences and Technology, Cambridge, MA 02139, USA
6. Bryn Mawr College, Bryn Mawr, PA
7. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA
8. These authors contributed equally
Biologists discover how pancreatic tumors lead to weight loss

Shortfall of digestive enzymes can lead to tissue breakdown in early stages of pancreatic cancer.

Anne Trafton | MIT News Office
June 20, 2018

Patients with pancreatic cancer usually experience significant weight loss, which can begin very early in the disease. A new study from MIT and Dana-Farber Cancer Institute offers insight into how this happens, and suggests that the weight loss may not necessarily affect patients’ survival.

In a study of mice, the researchers found that weight loss occurs due to a reduction in key pancreatic enzymes that normally help digest food. When the researchers treated these mice with replacement enzymes, they were surprised to find that while the mice did regain weight, they did not survive any longer than untreated mice.

Pancreatic cancer patients are sometimes given replacement enzymes to help them gain weight, but the new findings suggest that more study is needed to determine whether that actually benefits patients, says Matt Vander Heiden, an associate professor of biology at MIT and a member of the Koch Institute for Integrative Cancer Research.

“We have to be very careful not to draw medical advice from a mouse study and apply it to humans,” Vander Heiden says. “The study does raise the question of whether enzyme replacement is good or bad for patients, which needs to be studied in a clinical trial.”

Vander Heiden and Brian Wolpin, an associate professor of medicine at Harvard Medical School and Dana-Farber Cancer Institute, are the senior authors of the study, which appears in the June 20 issue of Nature. The paper’s lead authors are Laura Danai, a former MIT postdoc, and Ana Babic, an instructor in medicine at Dana-Farber.

Starvation mode

In a 2014 study, Vander Heiden and his colleagues found that muscle starts breaking down very early in pancreatic cancer patients, usually long before any other signs of the disease appear.

Still unknown was how this tissue wasting process occurs. One hypothesis was that pancreatic tumors overproduce some kind of signaling factor, such as a hormone, that circulates in the bloodstream and promotes breakdown of muscle and fat.

However, in their new study, the MIT and Dana-Farber researchers found that this was not the case. Instead, they discovered that even very tiny, early-stage pancreatic tumors can impair the production of key digestive enzymes. Mice with these early-stage tumors lost weight even though they ate the same amount of food as normal mice. These mice were unable to digest all of their food, so they went into a starvation mode where the body begins to break down other tissues, especially fat.

The researchers found that when they implanted pancreatic tumor cells elsewhere in the body, this weight loss did not occur. That suggests the tumor cells are not secreting a weight-loss factor that circulates in the bloodstream; instead, they only stimulate tissue wasting when they are in the pancreas.

The researchers then explored whether reversing this weight loss would improve survival. Treating the mice with pancreatic enzymes did reverse the weight loss. However, these mice actually survived for a shorter period of time than mice that had pancreatic tumors but did not receive the enzymes. That finding, while surprising, is consistent with studies in mice that have shown that calorie restriction can have a protective effect against cancer and other diseases.

“It turns out that this mechanism of tissue wasting is actually protective, at least for the mice, in the same way that limiting calories can be protective for mice,” Vander Heiden says.

Human connection

The intriguing findings from the mouse study prompted the research team to see if they could find any connection between weight loss and survival in human patients. In an analysis of medical records and blood samples from 782 patients, they found no link between degree of tissue wasting at the time of diagnosis and length of survival. That finding is important because it could reassure patients that weight loss does not necessarily mean that the patient will do worse, Vander Heiden says.

“Sometimes you can’t do anything about this weight loss, and this finding may mean that just because the patient is eating less and is losing weight, that doesn’t necessarily mean that they’re shortening their life,” he says.

The researchers say that more study is needed to determine if the same mechanism they discovered in mice is also occurring in human cancer patients. Because the mechanism they found is very specific to pancreatic tumors, it may differ from the underlying causes behind tissue wasting seen in other types of cancer and diseases such as HIV.

“From a mechanistic standpoint, this study reveals a very different way to think about what could be causing at least some weight loss in pancreatic cancer, suggesting that not all weight loss is the same across different cancers,” Vander Heiden says. “And it raises questions that we really need to study more, because some mechanisms may be protective and some mechanisms may be bad for you.”

Clary Clish, director of the Metabolomics Platform at the Broad Institute, and members of his research group also contributed to this work. The research was funded, in part, by the Lustgarten Foundation, a National Institutes of Health Ruth Kirschstein Fellowship, Stand Up 2 Cancer, the Ludwig Center for Molecular Oncology at MIT, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the MIT Center for Precision Cancer Medicine, and the National Institutes of Health.

Biologists discover function of gene linked to familial ALS

Study in worms reveals gene loss can lead to accumulation of waste products in cells.

Anne Trafton | MIT News Office
May 4, 2018

MIT biologists have discovered a function of a gene that is believed to account for up to 40 percent of all familial cases of amyotrophic lateral sclerosis (ALS). Studies of ALS patients have shown that an abnormally expanded region of DNA in a specific region of this gene can cause the disease.

In a study of the microscopic worm Caenorhabditis elegans, the researchers found that the gene has a key role in helping cells to remove waste products via structures known as lysosomes. When the gene is mutated, these unwanted substances build up inside cells. The researchers believe that if this also happens in neurons of human ALS patients, it could account for some of those patients’ symptoms.

“Our studies indicate what happens when the activities of such a gene are inhibited — defects in lysosomal function. Certain features of ALS are consistent with their being caused by defects in lysosomal function, such as inflammation,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, and the senior author of the study.

Mutations in this gene, known as C9orf72, have also been linked to another neurodegenerative brain disorder known as frontotemporal dementia (FTD), which is estimated to affect about 60,000 people in the United States.

“ALS and FTD are now thought to be aspects of the same disease, with different presentations. There are genes that when mutated cause only ALS, and others that cause only FTD, but there are a number of other genes in which mutations can cause either ALS or FTD or a mixture of the two,” says Anna Corrionero, an MIT postdoc and the lead author of the paper, which appears in the May 3 issue of the journal Current Biology.

Genetic link

Scientists have identified dozens of genes linked to familial ALS, which occurs when two or more family members suffer from the disease. Doctors believe that genetics may also be a factor in nonfamilial cases of the disease, which are much more common, accounting for 90 percent of cases.

Of all ALS-linked mutations identified so far, the C9orf72 mutation is the most prevalent, and it is also found in about 25 percent of frontotemporal dementia patients. The MIT team set out to study the gene’s function in C. elegans, which has an equivalent gene known as alfa-1.

In studies of worms that lack alfa-1, the researchers discovered that defects became apparent early in embryonic development. C. elegans embryos have a yolk that helps to sustain them before they hatch, and in embryos missing alfa-1, the researchers found “blobs” of yolk floating in the fluid surrounding the embryos.

This led the researchers to discover that the gene mutation was affecting the lysosomal degradation of yolk once it is absorbed into the cells. Lysosomes, which also remove cellular waste products, are cell structures which carry enzymes that can break down many kinds of molecules.

When lysosomes degrade their contents — such as yolk — they are reformed into tubular structures that split, after which they are able to degrade other materials. The MIT team found that in cells with the alfa-1 mutation and impaired lysosomal degradation, lysosomes were unable to reform and could not be used again, disrupting the cell’s waste removal process.

“It seems that lysosomes do not reform as they should, and material accumulates in the cells,” Corrionero says.

For C. elegans embryos, that meant that they could not properly absorb the nutrients found in yolk, which made it harder for them to survive under starvation conditions. The embryos that did survive appeared to be normal, the researchers say.

Neuronal effects

The researchers were able to partially reverse the effects of alfa-1 loss in the C. elegans embryos by expressing the human protein encoded by the c9orf72 gene. “This suggests that the worm and human proteins are performing the same molecular function,” Corrionero says.

If loss of C9orf72 affects lysosome function in human neurons, it could lead to a slow, gradual buildup of waste products in those cells. ALS usually affects cells of the motor cortex, which controls movement, and motor neurons in the spinal cord, while frontotemporal dementia affects the frontal areas of the brain’s cortex.

“If you cannot degrade things properly in cells that live for very long periods of time, like neurons, that might well affect the survival of the cells and lead to disease,” Corrionero says.

Many pharmaceutical companies are now researching drugs that would block the expression of the mutant C9orf72. The new study suggests certain possible side effects to watch for in studies of such drugs.

“If you generate drugs that decrease c9orf72 expression, you might cause problems in lysosomal homeostasis,” Corrionero says. “In developing any drug, you have to be careful to watch for possible side effects. Our observations suggest some things to look for in studying drugs that inhibit C9orf72 in ALS/FTD patients.”

The research was funded by an EMBO postdoctoral fellowship, an ALS Therapy Alliance grant, a gift from Rose and Douglas Barnard ’79 to the McGovern Institute, and a gift from the Halis Family Foundation to the MIT Aging Brain Initiative.