Research Area: Cell Biology

Measuring chromosome imbalance could clarify cancer prognosis

A study of prostate cancer finds “aneuploid” tumors are more likely to be lethal than tumors with normal chromosome numbers.

Anne Trafton | MIT News Office
May 13, 2019

Most human cells have 23 pairs of chromosomes. Any deviation from this number can be fatal for cells, and several genetic disorders, such as Down syndrome, are caused by abnormal numbers of chromosomes.

For decades, biologists have also known that cancer cells often have too few or too many copies of some chromosomes, a state known as aneuploidy. In a new study of prostate cancer, researchers have found that higher levels of aneuploidy lead to much greater lethality risk among patients.

The findings suggest a possible way to more accurately predict patients’ prognosis, and could be used to alert doctors which patients might need to be treated more aggressively, says Angelika Amon, the Kathleen and Curtis Marble Professor in Cancer Research in the Department of Biology and a member of the Koch Institute for Integrative Cancer Research.

“To me, the exciting opportunity here is the ability to inform treatment, because prostate cancer is such a prevalent cancer,” says Amon, who co-led this study with Lorelei Mucci, an associate professor of epidemiology at the Harvard T.H. Chan School of Public Health.

Konrad Stopsack, a research associate at Memorial Sloan Kettering Cancer Center, is the lead author of the paper, which appears in the Proceedings of the National Academy of Sciences the week of May 13. Charles Whittaker, a Koch Institute research scientist; Travis Gerke, a member of the Moffitt Cancer Center; Massimo Loda, chair of pathology and laboratory medicine at New York Presbyterian/Weill Cornell Medicine; and Philip Kantoff, chair of medicine at Memorial Sloan Kettering; are also authors of the study.

Better predictions

Aneuploidy occurs when cells make errors sorting their chromosomes during cell division. When aneuploidy occurs in embryonic cells, it is almost always fatal to the organism. For human embryos, extra copies of any chromosome are lethal, with the exceptions of chromosome 21, which produces Down syndrome; chromosomes 13 and 18, which lead to developmental disorders known as Patau and Edwards syndromes; and the X and Y sex chromosomes. Extra copies of the sex chromosomes can cause various disorders but are not usually lethal.

Most cancers also show very high prevalence of aneuploidy, which poses a paradox: Why does aneuploidy impair normal cells’ ability to survive, while aneuploid tumor cells are able to grow uncontrollably? There is evidence that aneuploidy makes cancer cells more aggressive, but it has been difficult to definitively demonstrate that link because in most types of cancer nearly all tumors are aneuploid, making it difficult to perform comparisons.

Prostate cancer is an ideal model to explore the link between aneuploidy and cancer aggressiveness, Amon says, because, unlike most other solid tumors, many prostate cancers (25 percent) are not aneuploid or have only a few altered chromosomes. This allows researchers to more easily assess the impact of aneuploidy on cancer progression.

What made the study possible was a collection of prostate tumor samples from the Health Professionals Follow-up Study and Physicians’ Health Study, run by the Harvard T.H. Chan School of Public Health over the course of more than 30 years. The researchers had genetic sequencing information for these samples, as well as data on whether and when their prostate cancer had spread to other organs and whether they had died from the disease.

Led by Stopsack, the researchers came up with a way to calculate the degree of aneuploidy of each sample, by comparing the genetic sequences of those samples with aneuploidy data from prostate genomes in The Cancer Genome Atlas. They could then correlate aneuploidy with patient outcomes, and they found that patients with a higher degree of aneuploidy were five times more likely to die from the disease. This was true even after accounting for differences in Gleason score, a measure of how much the patient’s cells resemble cancer cells or normal cells under a microscope, which is currently used by doctors to determine severity of disease.

The findings suggest that measuring aneuploidy could offer additional information for doctors who are deciding how to treat patients with prostate cancer, Amon says.

“Prostate cancer is terribly overdiagnosed and terribly overtreated,” she says. “So many people have radical prostatectomies, which has significant impact on people’s lives. On the other hand, thousands of men die from prostate cancer every year. Assessing aneuploidy could be an additional way of helping to inform risk stratification and treatment, especially among people who have tumors with high Gleason scores and are therefore at higher risk of dying from their cancer.”

“When you’re looking for prognostic factors, you want to find something that goes beyond known factors like Gleason score and PSA [prostate-specific antigen],” says Bruce Trock, a professor of urology at Johns Hopkins School of Medicine, who was not involved in the research. “If this kind of test could be done right after a prostatectomy, it could give physicians information to help them decide what might be the best treatment course.”

Amon is now working with researchers from the Harvard T.H. Chan School of Public Health to explore whether aneuploidy can be reliably measured from small biopsy samples.

Aneuploidy and cancer aggressiveness

The researchers found that the chromosomes that are most commonly aneuploid in prostate tumors are chromosomes 7 and 8. They are now trying to identify specific genes located on those chromosomes that might help cancer cells to survive and spread, and they are also studying why some prostate cancers have higher levels of aneuploidy than others.

“This research highlights the strengths of interdisciplinary, team science approaches to tackle outstanding questions in prostate cancer,” Mucci says. “We plan to translate these findings clinically in prostate biopsy specimens and experimentally to understand why aneuploidy occurs in prostate tumors.”

Another type of cancer where most patients have low levels of aneuploidy is thyroid cancer, so Amon now hopes to study whether thyroid cancer patients with higher levels of aneuploidy also have higher death rates.

“A very small proportion of thyroid tumors is highly aggressive and lethal, and I’m starting to wonder whether those are the ones that have some aneuploidy,” she says.

The research was funded by the Koch Institute Dana Farber/Harvard Cancer Center Bridge Project and by the National Institutes of Health, including the Koch Institute Support (core) Grant.

A new approach to targeting tumors and tracking their spread

Researchers develop nanosized antibodies that home in on the meshwork of proteins surrounding cancer cells.

Helen Knight | MIT News correspondent
May 6, 2019

The spread of malignant cells from an original tumor to other parts of the body, known as metastasis, is the main cause of cancer deaths worldwide.

Early detection of tumors and metastases could significantly improve cancer survival rates. However, predicting exactly when cancer cells will break away from the original tumor, and where in the body they will form new lesions, is extremely challenging.

There is therefore an urgent need to develop new methods to image, diagnose, and treat tumors, particularly early lesions and metastases.

In a paper published today in the Proceedings of the National Academy of Sciences, researchers at the Koch Institute for Integrative Cancer Research at MIT describe a new approach to targeting tumors and metastases.

Previous attempts to focus on the tumor cells themselves have typically proven unsuccessful, as the tendency of cancerous cells to mutate makes them unreliable targets.

Instead, the researchers decided to target structures surrounding the cells known as the extracellular matrix (ECM), according to Richard Hynes, the Daniel K. Ludwig Professor for Cancer Research at MIT. The research team also included lead author Noor Jailkhani, a postdoc in the Hynes Lab at the Koch Institute for Integrative Cancer Research.

The extracellular matrix, a meshwork of proteins surrounding both normal and cancer cells, is an important part of the microenvironment of tumor cells. By providing signals for their growth and survival, the matrix plays a significant role in tumor growth and progression.

When the researchers studied this microenvironment, they found certain proteins that are abundant in regions surrounding tumors and other disease sites, but absent from healthy tissues.

What’s more, unlike the tumor cells themselves, these ECM proteins do not mutate as the cancer progresses, Hynes says. “Targeting the ECM offers a better way to attack metastases than trying to prevent the tumor cells themselves from spreading in the first place, because they have usually already done that by the time the patient comes into the clinic,” Hynes says.

The researchers began developing a library of immune reagents designed to specifically target these ECM proteins, based on relatively tiny antibodies, or “nanobodies,” derived from alpacas. The idea was that if these nanobodies could be deployed in a cancer patient, they could potentially be imaged to reveal tumor cells’ locations, or even deliver payloads of drugs.

The researchers used nanobodies from alpacas because they are smaller than conventional antibodies. Specifically, unlike the antibodies produced by the immune systems of humans and other animals, which consist of two “heavy protein chains” and two “light chains,” antibodies from camelids such as alpacas contain just two copies of a single heavy chain.

Nanobodies derived from these heavy-chain-only antibodies comprise a single binding domain much smaller than conventional antibodies, Hynes says.

In this way nanobodies are able to penetrate more deeply into human tissue than conventional antibodies, and can be much more quickly cleared from the circulation following treatment.

To develop the nanobodies, the team first immunized alpacas with either a cocktail of ECM proteins, or ECM-enriched preparations from human patient samples of colorectal or breast cancer metastases.

They then extracted RNA from the alpacas’ blood cells, amplified the coding sequences of the nanobodies, and generated libraries from which they isolated specific anti-ECM nanobodies.

They demonstrated the effectiveness of the technique using a nanobody that targets a protein fragment called EIIIB, which is prevalent in many tumor ECMs.

When they injected nanobodies attached to radioisotopes into mice with cancer, and scanned the mice using noninvasive PET/CT imaging, a standard technique used clinically, they found that the tumors and metastases were clearly visible. In this way the nanobodies could be used to help image both tumors and metastases.

But the same technique could also be used to deliver therapeutic treatments to the tumor or metastasis, Hynes says. “We can couple almost anything we want to the nanobodies, including drugs, toxins or higher energy isotopes,” he says. “So, imaging is a proof of concept, and it is very useful, but more important is what it leads to, which is the ability to target tumors with therapeutics.”

The ECM also undergoes similar protein changes as a result of other diseases, including cardiovascular, inflammatory, and fibrotic disorders. As a result, the same technique could also be used to treat people with these diseases.

In a recent collaborative paper, also published in Proceedings of the National Academy of Sciences, the researchers demonstrated the effectiveness of the technique by using it to develop nanobody-based chimeric antigen receptor (CAR) T cells, designed to target solid tumors.

CAR T cell therapy has already proven successful in treating cancers of the blood, but it has been less effective in treating solid tumors.

By targeting the ECM of tumor cells, nanobody-based CAR T cells became concentrated in the microenvironment of tumors and successfully reduced their growth.

The ECM has been recognized to play crucial roles in cancer progression, but few diagnostic or therapeutic methods have been developed based on the special characteristics of cancer ECM, says Yibin Kang, a professor of molecular biology at Princeton University, who was not involved in the research.

“The work by Hynes and colleagues has broken new ground in this area and elegantly demonstrates the high sensitivity and specificity of a nanobody targeting a particular isoform of an ECM protein in cancer,” Kang says. “This discovery opens up the possibility for early detection of cancer and metastasis, sensitive monitoring of therapeutic response, and specific delivery of anticancer drugs to tumors.”

This work was supported by a Mazumdar-Shaw International Oncology Fellowship, fellowships for the Ludwig Center for Molecular Oncology Research at MIT, the Howard Hughes Medical Institute and a grant from the Department of Defence Breast Cancer Research Program, and imaged on instrumentation purchased with a gift from John S. ’61 and Cindy Reed.

The researchers are now planning to carry out further work to develop the nanobody technique for treating tumors and metastases.

The fluid that feeds tumor cells

The substance that bathes tumors in the body is quite different from the medium used to grow cancer cells in the lab, biologists report.

Anne Trafton | MIT News Office
April 16, 2019

Before being tested in animals or humans, most cancer drugs are evaluated in tumor cells grown in a lab dish. However, in recent years, there has been a growing realization that the environment in which these cells are grown does not accurately mimic the natural environment of a tumor, and that this discrepancy could produce inaccurate results.

In a new study, MIT biologists analyzed the composition of the interstitial fluid that normally surrounds pancreatic tumors, and found that its nutrient composition is different from that of the culture medium normally used to grow cancer cells. It also differs from blood, which feeds the interstitial fluid and removes waste products.

The findings suggest that growing cancer cells in a culture medium more similar to this fluid could help researchers better predict how experimental drugs will affect cancer cells, says Matthew Vander Heiden, an associate professor of biology at MIT and a member of the Koch Institute for Integrative Cancer Research.

“It’s kind of an obvious statement that the tumor environment is important, but I think in cancer research the pendulum had swung so far toward genes, people tended to forget that,” says Vander Heiden, one of the senior authors of the study.

Alex Muir, a former Koch Institute postdoc who is now an assistant professor at the University of Chicago, is also a senior author of the paper, which appears in the April 16 edition of the journal eLife. The lead author of the study is Mark Sullivan, an MIT graduate student.

Environment matters

Scientists have long known that cancer cells metabolize nutrients differently than most other cells. This alternative strategy helps them to generate the building blocks they need to continue growing and dividing, forming new cancer cells. In recent years, scientists have sought to develop drugs that interfere with these metabolic processes, and one such drug was approved to treat leukemia in 2017.

An important step in developing such drugs is to test them in cancer cells grown in a lab dish. The growth medium typically used to grow these cells includes carbon sources (such as glucose), nitrogen, and other nutrients. However, in the past few years, Vander Heiden’s lab has found that cancer cells grown in this medium respond differently to drugs than they do in mouse models of cancer.

David Sabatini, a member of the Whitehead Institute and professor of biology at MIT, has also found that drugs affect cancer cells differently if they are grown in a medium that resembles the nutrient composition of human plasma, instead of the traditional growth medium.

“That work, and similar results from a couple of other groups around the world, suggested that environment matters a lot,” Vander Heiden says. “It really was a wake up call for us that to really know how to find the dependencies of cancer, we have to get the environment right.”

To that end, the MIT team decided to investigate the composition of interstitial fluid, which bathes the tissue and carries nutrients that diffuse from blood flowing through the capillaries. Its composition is not identical to that of blood, and in tumors, it can be very different because tumors often have poor connections to the blood supply.

The researchers chose to focus on pancreatic cancer in part because it is known to be particularly nutrient-deprived. After isolating interstitial fluid from pancreatic tumors in mice, the researchers used mass spectrometry to measure the concentrations of more than 100 different nutrients, and discovered that the composition of the interstitial fluid is different from that of blood (and from that of the culture medium normally used to grow cells). Several of the nutrients that the researchers found to be depleted in tumor interstitial fluid are amino acids that are important for immune cell function, including arginine, tryptophan, and cystine.

Not all nutrients were depleted in the interstitial fluid — some were more plentiful, including the amino acids glycine and glutamate, which are known to be produced by some cancer cells.

Location, location, location

The researchers also compared tumors growing in the pancreas and the lungs and found that the composition of the interstitial fluid can vary based on tumors’ location in the body and at the site where the tumor originated. They also found slight differences between the fluid surrounding tumors that grew in the same location but had different genetic makeup; however, the genetic factors tested did not have as big an impact as the tumor location.

“That probably says that what determines what nutrients are in the environment is heavily driven by interactions between cancer cells and noncancer cells within the tumor,” Vander Heiden says.

Scientists have previously discovered that those noncancer cells, including supportive stromal cells and immune cells, can be recruited by cancer cells to help remake the environment around the tumor to promote cancer survival and spread.

Vander Heiden’s lab and other research groups are now working on developing a culture medium that would more closely mimic the composition of tumor interstitial fluid, so they can explore whether tumor cells grown in this environment could be used to generate more accurate predictions of how cancer drugs will affect cells in the body.

The research was funded by the National Institutes of Health, the Lustgarten Foundation, the MIT Center for Precision Cancer Medicine, Stand Up to Cancer, the Howard Hughes Medical Institute, and the Ludwig Center at MIT.

Life unfolding

Graduate student Marlis Denk-Lobnig investigates the biological forces that shape developing tissue to dictate form and function.

Raleigh McElvery
March 22, 2019

A few hours after fertilization, the fruit fly embryo is just a hollow sphere, slightly oblong in shape, until a band of cells on its surface furrows inward to form a new layer. This folding process takes only 15 minutes, but it’s critical for determining where the cells will go and what roles they will eventually play. In humans, errors in tissue folding can result in diseases like spina bifida, where the spine never fully closes.

Fourth-year graduate student Marlis Denk-Lobnig watches this gastrulation process occurring in fly embryos in real time, tagging molecules with fluorescent proteins to probe the forces that eventually shape a fully-formed organism. Every day, she gets to witness new life unfold — literally.

Denk-Lobnig spends most of her time with her eye to a microscope or generating genetic crosses in the “fly room” where she keeps her stocks — rows of tubes containing light brown insect food that emits an unmistakable odor, despite being corked with cotton swabs. Inside each neatly labeled container, scores of tiny flies mill around as they lay eggs and feast.

Given that her mother trained in chemistry and her father in physics, “it didn’t take much creativity to get into science early on.” Denk-Lobnig enjoyed physics throughout high school, but also maintained a keen interest in biology, which became more pronounced after she was diagnosed with an autoimmune disease affecting her thyroid and adrenal glands.

“In some ways, the question of how your own body works is the most tangible question to ask,” she says. “It’s fascinating to connect everyday experiences with mechanisms, and studying biology and medicine seemed like a powerful way to have a direct impact on life.”

She majored in molecular medicine at Georg August University in Göttingen, Germany, located several hours from her childhood home in Heidelberg. Inspired by a summer internship with MIT Biology alum and Rockefeller professor Cori Bargmann PhD ’87, Denk-Lobnig centered her undergraduate thesis on the role glial cells play in disease.

She graduated after only three years, the typical duration in Germany, and spent the next several months traveling and applying to graduate schools. She also visited Nepal, where she taught visual and performing arts — and a bit of gymnastics — at a local boarding school.

When she began at MIT in 2015, Denk-Lobnig took the opportunity to blend her expertise in biology with a renewed enthusiasm for physics. Although she is a full-fledged member of the Department of Biology, she is simultaneously enrolled in the interdepartmental Biophysics Certificate Program.

“Not many people know that MIT has a thriving biophysics community,” she says. “It’s a mix of mechanical engineers, chemists, biologists, and physicists. There are specific course requirements, and we go on retreats and participate in seminars to share our research and discuss collaborations.”

As a member of Adam Martin’s lab, Denk-Lobnig studies the cellular forces that shape tissue form and function. Martin is also affiliated with the certificate program, and was one of the faculty members who initially interviewed Denk-Lobnig for the graduate program.

“Biophysics is all about finding elegant explanations for everyday phenomena, and I really enjoy thinking about physical principles and how they apply to biological problems,” Denk-Lobnig says. “The methods we use in the Martin lab are also incredibly visual. You can literally see a fruit fly embryo fold, and watch as a sheet of cells furrows inside the embryo to form a second layer, which is important for development. It’s both informative and aesthetically pleasing.”

Denk-Lobnig began by focusing on a single molecule called Cumberland-GAP (C-GAP), which regulates one of the many proteins in charge of tissue folding: myosin. Myosin is responsible for muscle contraction, among other duties. With its characteristic forked shape — two “heads” protruding from string-like “tail” domain — myosin can appear to walk along the cell’s scaffolding, sometimes transporting cargo. Denk-Lobnig, though, is most interested in myosin’s ability to pull on developing tissue and create a fold.

Right before graduating, one of Denk-Lobnig’s former labmates noticed that depleting C-GAP seemed to alter the concentration (or “gradient”) of myosin across the tissue. Since this finding pertained to the very regulator she was studying, it piqued Denk-Lobnig’s interest. She wanted to know how molecules like C-GAP might influence myosin and impact folding, and her scope widened from the molecular level to include the entire tissue.

It’s unlikely, she says, that myosin is pulling with equal force across the tissue — “that wouldn’t constrict the sheet of cells very efficiently.” Instead, there’s probably more myosin in middle and less towards the edges, which contracts the cells in the middle of the sheet to a greater degree and creates the curvature that forms the crease of the fold. In the fruit fly, gastrulation occurs just three hours after the eggs are laid. Because the folding happens at the surface of the embryo, there’s no need for dissection to witness the entire event through a microscope.

Denk-Lobnig has begun exploring other regulators besides C-GAP to analyze their effects on the myosin gradient and cell curvature. She was one of the first members of the lab to introduce CRISPR-Cas9 into their testing protocol, and is currently the only one experimenting with optogenetic techniques. She also regularly participates in the lab book club, which features classics like The Bell Jar and One Hundred Years of Solitude.

Outside of lab, Denk-Lobnig serves as the president of MIT’s women’s club gymnastics team, volunteers to help run weekly Gymnastics Special Olympics events, and sings in a graduate student choir. She is also a member of the department’s peer support program, bioREFs.

Long-term, she plans to stay in academia and delve further into physics-based methods, like modeling and coding. If she could find a project that’s just as visual as her current work in the Martin lab, “that would definitely be a plus.”

Posted 3.21.19
How tumors behave on acid

Acidic environment triggers genes that help cancer cells metastasize.

Anne Trafton | MIT News Office
March 21, 2019

Scientists have long known that tumors have many pockets of high acidity, usually found deep within the tumor where little oxygen is available. However, a new study from MIT researchers has found that tumor surfaces are also highly acidic, and that this acidity helps tumors to become more invasive and metastatic.

The study found that the acidic environment helps tumor cells to produce proteins that make them more aggressive. The researchers also showed that they could reverse this process in mice by making the tumor environment less acidic.

“Our findings reinforce the view that tumor acidification is an important driver of aggressive tumor phenotypes, and it indicates that methods that target this acidity could be of value therapeutically,” says Frank Gertler, an MIT professor of biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and the senior author of the study.

Former MIT postdoc Nazanin Rohani is the lead author of the study, which appears in the journal Cancer Research.

Mapping acidity

Scientists usually attribute a tumor’s high acidity to the lack of oxygen, or hypoxia, that often occurs in tumors because they don’t have an adequate blood supply. However, until now, it has been difficult to precisely map tumor acidity and determine whether it overlaps with hypoxic regions.

In this study, the MIT team used a probe called pH (Low) Insertion Peptide (pHLIP), originally developed by researchers at the University of Rhode Island, to map the acidic regions of breast tumors in mice. This peptide is floppy at normal pH but becomes more stable at low, acidic pH. When this happens, the peptide can insert itself into cell membranes. This allows the researchers to determine which cells have been exposed to acidic conditions, by identifying cells that have been tagged with the peptide.

To their surprise, the researchers found that not only were cells in the oxygen-deprived interior of the tumor acidic, there were also acidic regions at the boundary of the tumor and the structural tissue that surrounds it, known as the stroma.

“There was a great deal of tumor tissue that did not have any hallmarks of hypoxia that was quite clearly exposed to acidosis,” Gertler says. “We started looking at that, and we realized hypoxia probably wouldn’t explain the majority of regions of the tumor that were acidic.”

Further investigation revealed that many of the cells at the tumor surface had shifted to a type of cell metabolism known as aerobic glycolysis. This process generates lactic acid as a byproduct, which could account for the high acidity, Gertler says. The researchers also discovered that in these acidic regions, cells had turned on gene expression programs associated with invasion and metastasis. Nearly 3,000 genes showed pH-dependent changes in activity, and close to 300 displayed changes in how the genes are assembled, or spliced.

“Tumor acidosis gives rise to the expression of molecules involved in cell invasion and migration. This reprogramming, which is an intracellular response to a drop in extracellular pH, gives the cancer cells the ability to survive under low-pH conditions and proliferate,” Rohani says.

Those activated genes include Mena, which codes for a protein that normally plays a key role in embryonic development. Gertler’s lab had previously discovered that in some tumors, Mena is spliced differently, producing an alternative form of the protein known as MenaINV (invasive). This protein helps cells to migrate into blood vessels and spread though the body.

Another key protein that undergoes alternative splicing in acidic conditions is CD44, which also helps tumor cells to become more aggressive and break through the extracellular tissues that normally surround them. This study marks the first time that acidity has been shown to trigger alternative splicing for these two genes.

Reducing acidity

The researchers then decided to study how these genes would respond to decreasing the acidity of the tumor microenvironment. To do that, they added sodium bicarbonate to the mice’s drinking water. This treatment reduced tumor acidity and shifted gene expression closer to the normal state. In other studies, sodium bicarbonate has also been shown to reduce metastasis in mouse models.

Sodium bicarbonate would not be a feasible cancer treatment because it is not well-tolerated by humans, but other approaches that lower acidity could be worth exploring, Gertler says. The expression of new alternative splicing genes in response to the acidic microenvironment of the tumor helps cells survive, so this phenomenon could be exploited to reverse those programs and perturb tumor growth and potentially metastasis.

“Other methods that would more focally target acidification could be of great value,” he says.

The research was funded by the Koch Institute Support (core) Grant from the National Cancer Institute, the Howard Hughes Medical Institute, the National Institutes of Health, the KI Quinquennial Cancer Research Fellowship, and MIT’s Undergraduate Research Opportunities Program.

Other authors of the paper include Liangliang Hao, a former MIT postdoc; Maria Alexis and Konstantin Krismer, MIT graduate students; Brian Joughin, a lead research modeler at the Koch Institute; Mira Moufarrej, a recent graduate of MIT; Anthony Soltis, a recent MIT PhD recipient; Douglas Lauffenburger, head of MIT’s Department of Biological Engineering; Michael Yaffe, a David H. Koch Professor of Science; Christopher Burge, an MIT professor of biology; and Sangeeta Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Technology and Electrical Engineering and Computer Science.

Pumping up red blood cell production
Greta Friar | Whitehead Institute
February 28, 2019

Cambridge, MA — Red blood cells are the most plentiful cell type in our blood and play a vital role transporting oxygen around our body and waste carbon dioxide to the lungs. Injuries that cause significant blood loss prod the body to secrete a one-two punch of signals – stress steroids and erythropoietin (EPO) – that stimulates red blood cell production in the bone marrow. These signals help immature cells along the path to becoming mature red blood cells. In a healthy individual, as much as half of their blood volume can be replenished within a week. Despite its importance, scientists are still working to unravel many aspects of red blood cell production. In a paper published online February 28 in the journal Developmental Cell, Whitehead Institute researchers describe work that refines our understanding of how stress steroids, in particular glucocorticoids, increase red blood cell production and how early red blood cell progenitors progress to the next stage of maturation toward mature red blood cells.

These findings are especially important for patients with certain types of anemia that do not respond to clinical use of EPO to stimulate the final stages of red cell formation, such as Diamond-Blackfan anemia (DBA). In this rare genetic disorder usually diagnosed in infants and toddlers, the bone marrow does not produce enough of early red blood cell progenitors, called burst forming unit-erythroids (BFU-Es), that respond to glucocorticoids. In both healthy people and DBA patients, these BFU-Es divide several times and mature before developing into colony forming unit-erythroids (CFU-Es) that that, stimulated by EPO, repeatedly divide and produce immature red blood cells that are released from the bone marrow into the blood. But the lack of BFU-Es in DBA patients means that the glucocorticoid signal has a limited target, and the cascade of cell divisions that should result in plentiful red blood cells is contracted and instead produces an insufficient amount.

One of the standard treatments for DBA is boosting red blood cell production with high doses of synthetic glucocorticoids, such as prednisone or prednisolone. But the mechanisms behind these drugs and their normal counterparts are not well understood. By deciphering the mechanisms by which glucocorticoids stimulate red cell formation, scientists may be able identify other ways to stoke CFU-E production – and ultimately red blood cell production – without synthetic glucocorticoids and the harsh side effects that their long-term use can cause, such as poor growth in children, brittle bones, muscle weakness, diabetes, and eye problems.

For more than two decades, Whitehead Institute Founding Member Harvey Lodish, has investigated glucocorticoids’ effects on red blood cell production. In his lab’s most recent paper, co-first authors and postdocs Hojun Li and Anirudh Natarajan, describe their research, which helps decipher how BFU-Es progress through their maturation process.

For more than 30 years, scientists have thought that glucocorticoids bestowed BFU-Es with a stem cell-like ability to divide until an unknown switch flipped and the cells matured to the CFU-E stage. By looking at gene expression in individual BFU-Es from normal mice, Li and Natarajan determined that the developmental progression from BFU-E to CFU-E is instead a smooth continuum. They also found that in mice glucocorticoids exert the greatest effect on the BFU-Es at the beginning of the developmental continuum by slowing their developmental progression without affecting their cell division rate. In other words glucocorticoids are able to effectively compensate for a decreased number of BFU-Es by allowing those that do exist, while still immature, to divide more times, producing in mice up to 14 times more CFU-Es than BFU-Es lacking exposure to glucocorticoids.

Li and Natarajan’s work reveals previously unknown aspects of the mechanism by which glucocorticoids stimulate red blood cell production. With this better understanding, scientists are one step closer toward pinpointing more targeted approaches to treat certain anemias such as DBA.

This work was supported by the National Institutes of Health (NIH grants DK06834813 and HL032262-25) and the American Society of Hematology and was performed with the assistance of Whitehead Institute’s Fluorescence Activated Cell Scanning (FACS) Facility and Genome Technology Core facility. Styliani Markoulaki, head of the Whitehead Genetically Engineered Models Center, and M. Inmaculada Barrasa of Bioinformatics and Research Computing (BaRC) are also co-authors of the paper.

 

Written by Nicole Giese Rura

***

Harvey Lodish’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 and a professor of biological engineering at Massachusetts Institute of Technology (MIT). Lodish serves as a paid consultant and owns equity in Rubius, a biotech company that seeks to exploit the use of modified red blood cells for therapeutic applications.

***

Citation:

“Rate of Progression through a Continuum of Transit-Amplifying Progenitor Cell States Regulates Blood Cell Production”

Developmental Cell, online February 28, 2019, https://doi.org/10.1016/j.devcel.2019.01.026

Hojun Li*, Anirudh Natarajan*, Jideofor Ezike, M. Inmaculada Barrasa, Yenthanh Le, Zoë A. Feder, Huan Yang, Clement Ma, Styliani Markoulaki, and Harvey F. Lodish.

*These authors contributed equally

Why too much DNA repair can injure tissue

Overactive repair system promotes cell death following DNA damage by certain toxins, study shows.

Anne Trafton | MIT News Office
February 14, 2019

DNA-repair enzymes help cells survive damage to their genomes, which arises as a normal byproduct of cell activity and can also be caused by environmental toxins. However, in certain situations, DNA repair can become harmful to cells, provoking an inflammatory response that produces severe tissue damage.

MIT Professor Leona Samson has now determined that inflammation is a key component of the way this damage occurs in photoreceptor cells in the retinas of mice. About 10 years ago, she and her colleagues discovered that overactive initiation of DNA-repair systems can lead to retinal damage and blindness in mice. The key enzyme in this process, known as Aag glycosylase, can also cause harm in other tissues when it becomes hyperactive.

“It’s another case where despite the fact that inflammation is there to protect you, in some circumstances it can actually be harmful, when it’s overactive,” says Samson, a professor emerita of biology and biological engineering and the senior author of the study.

Aag glycosylase helps to repair DNA damage caused by a class of drugs known as alkylating agents, which are commonly used as chemotherapy drugs and are also found in pollutants such as tobacco smoke and fuel exhaust. Retinal damage from these drugs has not been seen in human patients, but alkylating agents may produce similar damage in other human tissues, Samson says. The new study, which reveals how Aag overactivity leads to cell death, suggest possible targets for drugs that could prevent such damage.

Mariacarmela Allocca, a former MIT postdoc, is the lead author of the study, which appears in the Feb. 12 issue of Science Signaling. MIT technical assistant Joshua Corrigan, former postdoc Aprotim Mazumder, and former technical assistant Kimberly Fake are also authors of the paper.

A vicious cycle

In a 2009 study, Samson and her colleagues found that a relatively low level of exposure to an alkylating agent led to very high rates of retinal damage in mice. Alkylating agents produce specific types of DNA damage, and Aag glycosylase normally initiates repair of such damage. However, in certain types of cells that have higher levels of Aag, such as mouse photoreceptors, the enzyme’s overactivity sets off a chain of events that eventually leads to cell death.

In the new study, the researchers wanted to find exactly out how this happens. They knew that Aag was overactive in the affected cells, but they didn’t know exactly how it was leading to cell death or what type of cell death was occurring. The researchers initially suspected it was apoptosis, a type of programmed cell death in which a dying cell is gradually broken down and absorbed by other cells.

However, they soon found evidence that another type of cell death called necrosis accounts for most of the damage. When Aag begins trying to repair the DNA damage caused by the alkylating agent, it cuts out so many damaged DNA bases that it hyperactivates an enzyme called PARP, which induces necrosis. During this type of cell death, cells break apart and spill out their contents, which alerts the immune system that something is wrong.

One of the proteins secreted by the dying cells, known as HMGB1, stimulates production of chemicals that attract immune cells called macrophages, which specifically penetrate the photoreceptor layer of the retina. These macrophages produce highly reactive oxygen species — molecules that create more damage and make the environment even more inflammatory. This in turn causes more DNA damage, which is  recognized by Aag.

“That makes the situation worse, because the Aag glycosylase will act on the lesions produced from the inflammation, so you get a vicious cycle, and the DNA repair drives more and more degeneration and necrosis in the photoreceptor layer,” Samson says.

None of this happens in mice that lack Aag or PARP, and it does not occur in other cells of the eye or in most other body tissues.

“It amazes me how segmented this is. The other cells in the retina are not affected at all, and they must experience the same amount of DNA damage. So, one possibility is maybe they don’t express Aag, while the  photoreceptor cells do,” Samson says.

“These molecular studies are exciting, as they have helped define the underlying pathophysiology associated with retinal damage,” says Ben Van Houten, a professor of pharmacology and chemical biology at the University of Pittsburgh, who was not involved in the study. “DNA repair is essential for the faithful inheritance of a cell’s genetic material. However, the very action of some DNA repair enzymes can result in the production of toxic intermediates that exacerbate exposures to genotoxic agents.”

Varying effects

The researchers also found that retinal inflammation and necrosis were more severe in male mice than in female mice. They suspect that estrogen, which can interfere with PARP activity, may help to suppress the pathway that leads to inflammation and cell death.

Samson’s lab has previously found that Aag activity can also exacerbate damage to the brain during a stroke, in mice. The same study revealed that Aag activity also worsens inflammation and tissue damage in the liver and kidney following oxygen deprivation. Aag-driven cell death has also been seen in the mouse cerebellum and some pancreatic and bone marrow cells.

The effects of Aag overactivity have been little studied in humans, but there is evidence that healthy individuals have widely varying levels of the enzyme, suggesting that it could have different effects in different people.

“Presumably there are some cell types in the human body that would respond the same way as the mouse photoreceptors,” Samson says. “They may just not be the same set of cells.”

The research was funded by the National Institutes of Health.

Biologists answer fundamental question about cell size

The need to produce just the right amount of protein is behind the striking uniformity of sizes.

Anne Trafton | MIT News Office
February 7, 2019

MIT biologists have discovered the answer to a fundamental biological question: Why are cells of a given type all the same size?

In humans, cell size can vary more than 100-fold, ranging from tiny red blood cells to large neurons. However, within each cell type, there is very little deviation from a standard size. In studies of yeast, MIT researchers grew cells to 10 times their normal size and found that their DNA could not keep up with the demands of producing enough protein to maintain normal cell functions.

Furthermore, the researchers found that this protein shortage leads the cells into a nondividing state known as senescence, suggesting a possible explanation for how cells become senescent as they age.

“There are so many hypotheses out there that try to explain why senescence happens, and I think this data provides a beautiful and simple explanation for senescence,” says Angelika Amon, the Kathleen and Curtis Marble Professor in Cancer Research in the Department of Biology and a member of the Koch Institute for Integrative Cancer Research.

Amon is the senior author of the study, which appears in the Feb. 7 online edition of Cell. Gabriel Neurohr, an MIT postdoc, is the lead author of the paper.

Excessive size

To explore why cell size is so tightly controlled, the researchers prevented yeast cells from dividing by modifying a gene critical for cell division, so that it could be turned off at a certain temperature. These cells continued to grow, but they could not divide and they did not replicate their DNA.

The researchers discovered that as the cells expanded, their DNA and their protein-building machinery could not keep pace with the needs of such a large cell. This failure to produce enough protein led to the dilution of the cytoplasm and disruption of cell division. The researchers believe that many other fundamental cell processes that rely on cellular molecules finding and interacting with each other may also be impaired when cells are too big.

“Theoretical models predict that diluting the cytoplasm will decrease reaction rates. Every chemical reaction would occur more slowly, and some threshold concentrations of certain proteins may not be reached, so certain reactions would never happen because the concentrations are lower,” Neurohr says.

The researchers showed that yeast cells with two sets of chromosomes were able to grow to twice the size of yeast cells with just one set of chromosomes before becoming senescent, suggesting that the amount of DNA in the cells is the limiting factor in the cells’ ability to grow.

Experiments with human cells yielded similar results: In a study of human fibroblast cells, the researchers found that forcing the cells to grow to excessive sizes (eight times their normal size) disrupted many functions, including cell division.

“It’s been clear for some time that cells do control their size, but it’s been unclear what the various physiological reasons are for why they do so,” says Jan Skotheim, an associate professor of biology at Stanford University, who was not involved in the research. “What’s nice about this work is it really shows how things go wrong when cells get too big.”

Age-related disease

Amon says excessive growth likely plays a major role in the development of senescence, which occurs in many types of mammalian cells and is thought to contribute to age-related organ dysfunction and chronic age-related diseases.

Senescent cells are often larger than younger cells, and this study, which showed that unchecked cell growth leads to senescence, offers a possible explanation for this observation. Human cells tend to grow slightly larger throughout their lifetimes, because every time a cell divides, it checks for DNA damage, and if any is found, division is halted while repairs are made. During each of these delays, the cell grows slightly larger.

“Over the lifetime of a cell, the more divisions you make, the higher your probability is of having that damage, and over time cells will get larger,” Amon says. “Eventually they get so large that they start diluting critical factors that are important for proliferation.”

A difficult question that remains unanswered is how different types of cells maintain the appropriate size for their cell type, which the researchers now hope to study further.

The research was funded, in part, by the National Institutes of Health, the Howard Hughes Medical Institute, the Paul F. Glenn Center for Biology of Aging Research at MIT, a National Science Foundation graduate research fellowship, the William Bowes Fellows program, and the Vilcek Foundation.

Origin story

Junior Leah McKinney practiced kitchen microbiology on her ranch in Nevada before exploring the intricacies of DNA replication initiation in bacteria at MIT Biology.

Raleigh McElvery
February 6, 2019

Leah McKinney grew up on a 50,000-acre cattle ranch in Nevada — vaccinating sheep, roping calves, digging for fossils, and occasionally hauling home old bovine femurs. She saddled horses, treated sick lambs, and helped ewes struggling to give birth. One Christmas, she even asked Santa for a fetal pig. “He delivered,” McKinney, now a junior in Course 7, recalls with a laugh.

When she was 12 years old, she saved up enough birthday money to purchase a microscope. Even though she permanently dyed the kitchen sink a distinct shade of blue while making slides, her parents (who both hold degrees in animal science) didn’t mind. They even let her grow bacteria in the heater closet and tally them on the kitchen counter — all in the name of the elementary school science fair.

“They were always encouraging my weird scientific endeavors,” she says. “I think my love for science, and microbiology specifically, came out of my agricultural upbringing.”

She grew to appreciate basic science because it allowed her to study the fundamental mechanisms behind key biological processes. She arrived at MIT in 2016 determined to major in Biology, and hasn’t wavered in her decision. Although she relishes the subject matter, she initially feared the classes would be tedious and memorization-heavy.

“I was quite happy to learn that’s not the case here,” she says. “MIT Biology values problem-solving over rote memorization, and encourages you to take the information you’ve learned in class and apply it to interesting problems. And that mindset extends from the classroom into the lab.”

One of the things that drew McKinney to MIT was the institute’s Undergraduate Research Opportunities Program (UROP), which allows students to join labs and collaborate with faculty as early as their first year. She recalls that, while other universities touted similar opportunities, MIT placed theirs front and center.

“I’d heard that all you had to do was email a professor and ask to join the lab, but I didn’t believe it — that just seemed way too easy,” McKinney says. “But when I was looking for a UROP, I just emailed my current principal investigator to set up a time to talk, and now I’ve been in his lab for over a year.”

McKinney is part of Department Head Alan Grossman’s lab, which investigates the molecular mechanisms and regulation underlying basic cellular processes in bacteria. The entire group works with the rod-shaped Bacillus subtilis, but some members study horizontal gene transfer while others focus on DNA replication and gene expression. McKinney and her graduate student mentor Mary Anderson are in this second category, examining a protein called DnaA that is required to initiate DNA replication and also modulates the expression of several genes.

In order to successfully grow and reproduce, a bacterium must first replicate its single chromosome before dividing into two identical daughter cells. DnaA is responsible for beginning DNA replication in all bacteria. It binds to the origin of replication on the chromosome, unwinds some of the nearby DNA, and recruits the other proteins needed to copy the chromosome.

This operation is highly regulated to ensure that each daughter cell receives only a single chromosome. B. subtilis controls replication via several proteins, including YabA. When YabA binds to DnaA, it prevents replication from ever getting started.

Since DnaA also serves as a transcription factor — binding to other DNA sequences called promoters to increase or decrease expression of certain genes, including its own gene dnaA — YabA may also impact DnaA’s gene targets. McKinney hopes to eventually determine exactly how.

While McKinney discovers something new about her bacteria each time she conducts a successful experiment, she learns almost as much when her tests go awry. “I’ve had to practice a lot of troubleshooting,” she says, “and that’s not something you can learn in class. But everyone in the lab is incredibly friendly and always willing to answer questions or give advice.”

As a teaching assistant for the lab class 7.02 (Introduction to Experimental Biology and Communication), McKinney had the chance to help other students conduct experiments, answering their questions and grading their lab notebooks. She took 7.02 last spring, but says it’s been enlightening to experience the class through a different lens. She adds: “I definitely understand the material more deeply than I did before.”

In addition to TAing, McKinney teaches an SAT preparatory program run by MIT students. “At first, standing up and talking in front of a 20-person section was rather terrifying, but it’s become so much easier,” she says. “The experience has been really good for me.”

After she graduates, McKinney knows she wants to go to graduate school — likely for microbiology — but beyond that, nothing is concrete. She is sure of one thing, though: joining the Grossman lab was one of the best decisions she’s made at MIT.

She advises all current and prospective students to do a UROP. “Find something you’re really interested in,” she says. “It’s okay not to know a lot coming in; you’re going to learn so much, including topics and techniques you won’t learn in class. And don’t be too disappointed when things don’t work; that’s just part of the process. And when you finally get something to work that you’ve been troubleshooting for a while, the feeling is absolutely amazing.”

Posted 2.5.19