Intermountain Healthcare Researchers Launch Major Three-Year Genomics Breast Cancer Study

Cancer researchers at Intermountain Medical Center and the Intermountain Healthcare Precision Genomics Program in Salt Lake City are launching a three-year study to determine if a blood test that looks for DNA from a cancer tumor can be used to complement mammography to improve the way breast cancer is diagnosed.

The goal of this new genomics study is to show whether screening patients for the presence of circulating tumor DNA, known as ctDNA, can successfully detect breast cancer using a blood draw.

Breast cancer is the second-leading cause of cancer deaths in women, behind only lung cancer, with an estimated 40,610 deaths each year from the disease. Nearly 253,000 new cases of invasive breast cancer are diagnosed each year, along with about 60,000 non-invasive, early-stage cases, according to the American Cancer Society.

The Intermountain study is unique in that researchers will also help develop a specific test to check for ctDNA, and will have access to both mammography results and the DNA blood test results, which will allow a direct comparison of the “liquid-based biopsy” to be made.

The idea behind the science is simple, though researchers say the execution is not yet proven: Little pieces of DNA that come from dying cells end up in the peripheral blood stream, including circulating tumor cells. The goal of researchers is to use those markers to identify breast cancer, perhaps even before mammography can detect it, said Lincoln Nadauld, MD, PhD, co-lead investigator of the study and executive director of the Intermountain Healthcare Precision Genomics Program.

“As a tumor is growing, some of the cells will die and their DNA will end up in the peripheral blood stream,” Dr. Nadauld said. “We’re able to distinguish DNA from cancer vs. DNA from normal cells. The idea is to leverage DNA to see if we can detect that it comes from a tumor.”

In the study, patients with known breast cancer will be compared with those in a screening group.

“We don’t know what we’ll see yet,” said Brett Parkinson, MD, co-lead investigator of the study, who is also imaging director and medical director of the Intermountain Medical Center Breast Care Center in Murray. “We might find those who have breast cancer will have a negative blood test and learn it’s not a good screening tool.”

Even a successful blood test isn’t expected to replace mammography outright. If it detects the circulating tumor DNA, imaging would be needed to find the tumor. But it could help eliminate unneeded biopsies, Dr. Parkinson added.

Dr. Nadauld said cancers have mutations in their DNA that aren’t always unique.

“Sometimes those are the same whether it’s a breast cancer or a colon cancer. If we do create a blood test, it’s possible it would detect mutant DNA, but it might look so similar it would be hard to tell what kind of cancer it came from,” he said. “That’s part of what this trial is going to accomplish. We want to determine the signature for early breast cancer.”

If successful, a liquid biopsy might also be used to monitor a breast cancer survivor for recurrence, Dr. Nadauld said. It might even lead to development of similar tests for different types of cancer. But that would be a challenge for the future.

“We want to approach this with laser-like focus,” he said. “It’s needed to help us diagnose breast cancer. We need to detect it earlier, when it’s curable.”

Breast cancer survival depends largely on finding the disease early —and mammography is the only screening exam that’s been shown by multiple randomized clinical trials to reduce the mortality rate for breast cancer. Since 1991, the death rate from breast cancer is down 38 percent, largely because mammography screening tests lead to early detection.

Although mammography finds most breast cancers, it may not detect malignancy in women who have dense breast tissue, especially premenopausal women, or those under 50.

“We pick up most breast cancer in women with average breast density,” said Dr. Parkinson. “When breast tissue is denser, we can miss up to 30 percent of breast cancers.”

Mammography also has a false-positive or call-back rate of 10 percent, which may subject women to additional imaging and emotional duress. Plus, a mammogram can be uncomfortable, since breast tissue is compressed for imaging, which also exposes a woman to a small amount of radiation. Mammography may also be inconvenient, often requiring women to take time off work, he noted.

For those, and perhaps other reasons, mammography screening rates in the United States are low. In Utah, only about 65 percent of eligible women are screened, despite Intermountain Healthcare’s recommendations that women over 40 undergo yearly screening mammography. All major medical and advocacy organizations agree that screening every year after a woman is 40 saves more lives. About 20 percent of breast cancers occur in women under 50.

Dr. Nadauld said the unusual confluence of three factors weigh in Intermountain’s favor on this quest, starting with access to a lot of patients in one place who are getting mammograms, which are the gold standard screening test for breast cancer. Second, the researchers have access to the results of those mammograms; they know if the results were positive or negative. The third major factor is Intermountain’s genomic technology capability.

“This is the big conversation right now in all of oncology — the use of liquid biopsy to determine how to screen for breast cancer, a woman’s risk of recurrence, and how to monitor their treatment,” Dr. Nadauld said.

The study is being made possible by a generous donation from the Beesley Family Foundation.

Tiny Nanopackages Built Out of DNA Help Scientists Peek at How Neurons Work

A team of scientists from the University of Chicago designed a way to use microscopic capsules made out of DNA to deliver a payload of tiny molecules directly into a cell. The technique, detailed Aug. 21 in Nature Nanotechnology, gives scientists an opportunity to understand certain interactions among cells that have previously been hard to track.

“It’s really a molecular platform,” said Yamuna Krishnan, professor in chemistry and co-author of the study (pictured above). “There are a host of research problems from cardiology to neurobiology that need a system like this to study very fast molecular phenomena, so it could be applied in a variety of ways.”

Cells talk to each other in chemical whispers that occur too fast for scientists to accurately study, Krishnan said. Her team aimed at one class of such chemical communications, known as neurosteroids.

Scientists know neurosteroids are involved in neuronal health, but they’re difficult to study because they operate on hair triggers. “The moment you add a neurosteroid, the neuron’s already fired,” Krishnan said.

Researchers want a blow-by-blow account of what happens in the cell as the neurosteroid plays its part in the intricate signaling dance inside a neuron. To do so, they needed to get the neurosteroids to the cell inside a little package, release them on cue and then track what happens. But it’s difficult to make a delivery system so airtight that it doesn’t leak a couple of molecules before everything’s set up.

For this task, Krishnan had a solution: Her lab builds tiny machines out of DNA. It’s a good material because like a set of Legos, it has standard interlocking pieces that make it easy to build into configurations. And since it’s made out of parts already in the body, it can dissolve harmlessly once its purpose is achieved.

The lab made tiny structures—icosahedral, like a 20-sided die—with two halves that clamp together around a payload of molecules to form a capsule. Each capsule is just 20 nanometers across; that’s a thousand times smaller than the width of a human hair.

The next step was to send them to key locations inside the body by finding the right molecular “addresses” to particular cells, and gluing them onto the capsule. (Scientists find these addresses by studying how viruses and bacteria hone in on particular parts of the body.) To release the payload from the capsule, the scientists simply shine a light on the cells.

They tested and confirmed the system in worms and were able to measure the kinetics of the neurosteroids, previously an elusive process, the authors said.

Someday, Krishnan said, the technology could be used to deliver drugs or treatment to certain parts of the body, but theirs was a case study to explore the method as a way to better understand our own bodies and how they work.

URI Scientist: Rare Childhood Disease Linked to Major Cancer Gene

Researcher discovers novel connection between Fanconi anemia, PTEN

A team of researchers led by a University of Rhode Island scientist has discovered an important molecular link between a rare  childhood genetic disease, Fanconi anemia, and a major cancer gene called PTEN. The discovery improves the understanding of the molecular basis of Fanconi anemia and could lead to improved treatment outcomes for some cancer patients.

According to Niall Howlett, URI associate professor of cell and molecular biology and Rhode Island’s leading expert on Fanconi anemia, the disease is characterized by birth defects, bone marrow failure and increased cancer risk. He said the genes that play a role in the development of the disease are also important in the development of hereditary breast and ovarian cancer.

Howlett’s new study now establishes a molecular link between Fanconi anemia and a gene strongly associated with uterine, prostate and brain cancer. This research was published this month in the journal Scientific Reports, with URI graduate student Elizabeth Vuono as lead author.

About 1 in 150,000 children in the United States is born with Fanconi anemia.

“People often ask why we study such a rare disease,” said Howlett, who has been studying Fanconi anemia for nearly 20 years. “First and foremost, there is no cure or effective treatments for it. So a greater understanding of the molecular basis of Fanconi anemia is critical to address this need.”

In addition, Howlett said there are countless examples of how the study of Fanconi anemia has greatly benefited the general population. The first umbilical cord blood transplant, for example, was performed with a Fanconi anemia patient. Bone marrow transplants have become much safer and more effective because of studies with Fanconi anemia patients. And new breast and ovarian cancer genes have been discovered as a result of studies on the molecular biology of Fanconi anemia.

Howlett’s current research is another example of the broader impact of Fanconi anemia studies.

The URI researcher speculated about the existence of a biochemical link between Fanconi anemia and PTEN. Mutations in PTEN occur frequently in uterine, prostate and brain cancer.

“The PTEN gene codes for a phosphatase – an enzyme that removes phosphate groups from proteins,” explained Howlett. “Many Fanconi anemia proteins have phosphate groups attached to them when they become activated. However, how these phosphate groups are removed is poorly understood.”

Howlett said that cells from Fanconi anemia patients are characteristically sensitive to a class of drugs widely used in cancer chemotherapy called DNA crosslinking agents.

“So we performed an experiment to determine if Fanconi anemia and PTEN were biochemically linked,” he said. “By testing if cells with mutations in the PTEN gene were also sensitive to DNA crosslinking agents, we discovered that Fanconi anemia patient cells and PTEN-deficient cells were practically indistinguishable in terms of sensitivity to these drugs. This strongly suggested that the Fanconi anemia proteins and PTEN might work together to repair the DNA damage caused by DNA crosslinking agents.”

By using epistasis analysis, a genetic method that determines if genes work together, Howlett and his research group found that the Fanconi anemia proteins and PTEN do indeed function together in this repair pathway.

“Before this work, Fanconi anemia and PTEN weren’t even on the same radar,” said Howlett. “This is really important to understanding how this disease arises and what its molecular underpinnings are. The more we can find out about its molecular basis, the more likely we are to come up with strategies to treat the disease.”

Howlett’s research is equally important to cancer patients who do not have Fanconi anemia. He said that since his study found that cells missing PTEN are highly sensitive to DNA crosslinking agents, it should be possible to predict whether a particular cancer patient will respond to this class of chemotherapy drug by conducting a simple DNA test.

“We can now predict that if a patient has cancer associated with mutations in PTEN, then it is likely that the cancer will be sensitive to DNA crosslinking agents,” he said. “This could lead to improved outcomes for patients with certain types of PTEN mutations.”

LJI Scientists Flip Molecular Switches To Distinguish Closely Related Immune Cell Populations

The cornerstone of genetics is the loss-of-function experiment. In short, this means that to figure out what exactly gene X is doing in a tissue of interest—be it developing brain cells or a pancreatic tumor—you somehow cut out, switch off or otherwise destroy gene X in that tissue and then watch what happens. That genetic litmus test has been applied since before people even knew the chemical DNA is what makes up genes. What has changed radically are the tools used by biologists to inactivate a gene.

Until now, scientists wishing to delete a gene in a model organism like a mouse did it by clipping out stretches of DNA encoding entire genes or very big chunks of them from the animal’s genome. This type of gene “knockout” is what La Jolla Institute for Allergy and Immunology (LJI) investigator Catherine C. Hedrick, Ph.D., used in 2011, when her lab discovered that mice without the gene Nr4a1 lack an anti-inflammatory subtype of white blood cells, nicknamed ‘patrolling monocytes’.

Now, the Hedrick group’s latest study reports a next-generation molecular manipulation aimed at inactivating Nr4a1 in a more precise manner. That study, published in the November 15, 2016, edition of Immunity, reports the loss of the same patrolling monocyte population following inactivation of a molecular switch that turns on Nr4a1. “This new work is exciting, because it shows that we can directly target genes within a specific cell type, which is important for targeted therapies,” says Hedrick, a Professor in the Division of Inflammation Biology.

The Hedrick laboratory’s previous demonstration that patrolling monocytes disappear following global Nr4a1 loss proved that the gene is necessary for development of that cell type. Later, her group reported that cancer cells injected into mice lacking Nr4a1 (therefore lacking patrolling monocytes) underwent unchecked metastasis, supporting the idea that patrolling monocytes play anti-cancer roles. But an important experimental question lingered: could the cancer metastasis seen in Nr4a1 knockout mice have anything to do with potential loss of Nr4a1 in a closely related group of cells called macrophages, which use Nr4a1 to control inflammation?

The new paper answers this question by silencing Nr4a1 only in patrolling monocytes. The Hedrick group accomplished this by applying good old-fashioned biochemistry to isolate stretches of DNA that flank the gene and define the on-switch for monocytes. Scientists call tissue-specific gene regulatory elements like this “enhancers.” They then showed that when activated, that DNA region, which they called “enhancer #2” (E2), was capable of switching on Nr4a1 expression only in patrolling monocytes, and not in related cells like macrophages.

The group proved the specificity of the enhancer by engineering mice whose genomes lacked only the E2 enhancer—not the gene itself—and indeed observed a lack of patrolling monocytes. “Until now, we did not have a way to delete a gene only in monocytes without also deleting it in macrophages,” says Graham Thomas, Ph.D., a postdoc in the Hedrick lab and the study’s first author. “Targeting the enhancer allows us to study particular cell types in a highly specific way,” says Thomas. “Also, eliminating enhancers teaches us what turns these genes on in the first place. That knowledge is essential if we are going to design rational targets to go after these cells.”

To confirm that macrophages throw an entirely different molecular switch to turn on Nr4a1, the group exposed mice missing the monocyte E2 switch to a noxious toxin found in bacterial membranes, as a way of seeing whether macrophages can still mount normal inflammatory responses. Indeed, the macrophage response was entirely normal in E2 mutants, unlike the global Nr4a1 “knockout”, showing that macrophages do not use the genetic E2 switch.

Finally, to make sure that E2 enhancer loss mimicked deletion of the entire gene in monocytes the group revisited a tumor model previously used to test Nr4a1’s anti-cancer effect. To do so, they injected melanoma cells into the bloodstream of normal or E2 mutant mice and monitored lung metastasis. Remarkably, outcomes following loss of the switch mirrored what the group had previously observed when they physically removed the gene itself: the lungs of mutant mice contained many more melanoma cells than did lungs of normal mice. This confirmed that the gene regulatory switch is highly specific to one cell type, monocytes and that tumor cell invasion in the absence of this population had nothing to do with deregulated macrophage activity.

Hedrick also thinks the new findings provide new understanding of just how important DNA enhancer regions can be. “Being able to selectively target specific cell types opens up a new world for understanding how to design therapies to treat disease,” she says.

Researchers Restore Drug Sensitivity in Breast Cancer Tumors

A team of Case Western Reserve University School of Medicine cancer researchers has uncovered one way certain tumors resist vital medication.

In the study published in Oncotarget, the researchers studied tumor biopsies collected from breast cancer patients before and after treatment with the go-to breast cancer drug trastuzumab (also known as Herceptin). Some of the tumors were treatable with trastuzumab, and others were not. By comparing genes activated in the responsive tumors to those in non-responsive tumors, the researchers uncovered several genes that may help tumors evade the drug. Tumors previously resistant to trastuzumab were resensitized when the researchers blocked one of the genes, called S100P.

The study zeroed in on small pieces of genetic material called mRNAs and lincRNAs. These tiny fragments are created from DNA inside normal cells but become dysregulated in tumors. The research team initially identified 1,542 mRNAs and 371 lincRNAs that were different between tumors cells responsive to trastuzumab and non-responsive tumors. These differences indicated to the researchers that separate networks of cell signals were being activated in each group of tumor cells. The researchers narrowed down the list of RNAs using cells grown in their laboratory. They were interested in finding an RNA molecule that could be therapeutically manipulated to disrupt signals in the tumor cells related to trastuzumab resistance.

Ahmad Khalil, PhD, Assistant Professor of Genetics at Case Western Reserve University School of Medicine led the study and explained, “Our hypothesis was that there are gene expression differences of both mRNAs and lincRNAs between tumors from patients that respond to trastuzumab and tumors from patients that do not.”

Trastuzumab works by sticking to a protein called HER2 found on the surfaces of 25-30% of early-stage breast cancer tumor cells. The drug prevents HER2 from activating and controlling genes inside breast cancer cells. The research team grew breast cancer tumor cells with HER2 on their surfaces in their laboratory so they could validate findings from tumor biopsies. They exposed the cells to trastuzumab, mimicking cancer treatment regimens. Some breast cancer cells became resistant to trastuzumab after long-term exposure, just like the tumors collected from patients.

The researchers could identify mRNAs and lincRNAs that varied between trastuzumab-resistant and -sensitive HER2 cancer cells grown in the laboratory. They looked for overlap between the list of different RNAs in tumor biopsies and laboratory-grown cancer cells. The team identified 18 mRNAs and 7 lincRNAs that were associated with trastuzumab resistance in both models. The team zeroed in on a single gene that may be central to trastuzumab resistance after performing additional experiments in the laboratory.

The gene, S100P, is highly activated in breast cancer cells resistant to trastuzumab, as compared to normal breast tissue. Other studies have associated S100P with prostate and pancreatic cancers. It belongs to a family of genes that work together to support tumor growth and has been found in multiple compartments inside cancer cells.

“S100P was one of the key genes that showed significant expression differences,” said Khalil. “It further stood out because it was part of a pathway that emerged from a separate set of computational analyses of large datasets.”

The researchers designed small pieces of genetic material to block S100P in breast cancer cells. Cells grown in the laboratory that were previously resistant to trastuzumab became sensitive to the drug after exposure to S100P blockers. Further analyses indicated that S100P activates critical proteins inside breast cancer cells to compensate for those turned off when trastuzumab blocks HER2. The activated proteins may help tumor cells adjust their gene expression in response to drugs in their environment.

“Our data demonstrated that high expression levels of S100P are required for cancer cells to become resistant to trastuzumab,” concluded Khalil.

This exciting discovery indicates that depleting S100P in breast cancer may be one way to resensitize tumors to trastuzumab. The next step will be to further investigate the resistance mechanism, and screen for drugs that could be used to block S100P in human tumors. The researchers also plan to investigate the role of other mRNAs and lincRNAs from their list in regulating trastuzumab resistance.

Approximately one-third of early-stage breast cancer patients relapse after trastuzumab treatment, even if the drug is successful at first. Tumors in relapsed patients become resistant to trastuzumab which limits further treatment options. The mechanism behind trastuzumab resistance has not been easy to identify. Some studies have proposed mechanisms of trastuzumab resistance using cell culture models, but this study is the first to find mechanisms present in both cells growing in a laboratory dish and tumors growing in breast cancer patients.

According to Khalil, “Trastuzumab is a first line treatment for breast cancer patients with HER2 gene amplification. Thus, finding the mechanism of resistance to this major drug now opens the door to reverse the resistance, potentially curing many more patients.”

Scientists keep a molecule from moving inside nerve cells to prevent cell death

Amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease) is a progressive disorder that devastates motor nerve cells. People diagnosed with ALS slowly lose the ability to control muscle movement, and are ultimately unable to speak, eat, move, or breathe. The cellular mechanisms behind ALS are also found in certain types of dementia.

A groundbreaking scientific study published in Nature Medicine has found one way an RNA binding protein may contribute to ALS disease progression. Cells make RNA to carry instructions for making proteins from DNA to protein-constructing machinery.

The culprit protein, TDP-43, normally binds to small pieces of newly read RNA and helps shuttle the fragments around inside nerve cell nuclei. The study describes for the first time the molecular consequences of misplaced TDP-43 inside nerve cells, and demonstrates that correcting its location can restore nerve cell function. Misplacement of TDP-43 in nerve cells is a hallmark of ALS and other neurological disorders including frontotemporal dementia (FTD), Alzheimer’s, Parkinson’s, and Huntington’s diseases. Studies that characterize common mechanisms behind these diseases could have widespread implications and may also accelerate development of broad-based therapies.

To find the misplaced TDP-43, the researchers viewed nerve cells donated by people who died from ALS or FTD under high powered microscopes. They discovered TDP-43 accumulates in nerve cell mitochondria, critical structures responsible for generating the enormous amount of energy nerve cells require. By physically isolating the affected mitochondria the researchers were able to pinpoint TDP-43’s exact location inside the subcellular structures. They were also able to characterize variations of the protein most likely to get misplaced.

This important work was led by Xinglong Wang, PhD, from the department of pathology at Case Western Reserve University School of Medicine and a team of scientists from his laboratory.

“By multiple approaches, we have identified the mitochondrial inner membrane facing matrix as the major site for mitochondrial TDP-43,” explained Wang. “Mitochondria might be major accumulation sites of TDP-43 in dying neurons in various major neurodegenerative diseases.”

The researchers discovered that once inside the mitochondria, TDP-43 resumes its RNA binding role and attaches itself to mitochondrial genetic material. This disrupts the mitochondria’s ability to generate energy for the cell. Wang’s team was able to precisely identify the RNA in mitochondria that was bound by TDP-43 and observe the resultant disassembly of mitochondrial protein complexes. This finding provides much needed clarity on the consequences of TDP-43 misplacement inside nerve cells and opens the door for deeper studies involving a range of neurological disorders. Although the study focused on ALS and FTD, according to Wang “mislocalization of TDP-43 represents a key pathological feature correlating strongly with symptoms in more than half of Alzheimer’s disease patients.”

Mutations in the gene encoding TDP-43 have long been linked to neurodegenerative diseases like ALS and FTD. Wang’s team found that disease-associated mutations in TDP-43 enhance its misplacement inside nerve cells. The researchers also identified sections of TDP-43 that are recognized by mitochondria and serve as signals to let it inside. These sections could serve as therapeutic targets, as the study found blocking them prevents TDP-43 from localizing inside mitochondria. Importantly, Wang’s team was able to keep TDP-43 out of nerve cell mitochondria in mice using small proteins which “almost completely” prevented nerve cell toxicity and disease progression.

“We, for the first time, provide the novel concept that the inhibition of TDP-43 mitochondrial localization is sufficient to prevent TDP-43-linked neurodegeneration,” said Wang. “Targeting mitochondrial TDP-43 could be a novel therapeutic approach for ALS, FTD and other TDP-43-linked neurodegenerative diseases.”

Wang has begun to develop small proteins that prevent TDP-43 from reaching mitochondria in human nerve cells, and has a patent pending for the therapeutic molecule used in the study.

There is no treatment currently available for ALS or FTD. The average life expectancy for people newly diagnosed with ALS is just three years, according to The ALS Association.

 

Genetic Profiling Increases Cancer Treatment Options, Sanford Study Finds

Genetic profiling of cancer tumors provides new avenues for treatment of the disease, according to a study conducted by Sanford Health and recognized by the American Society of Clinical Oncology.

In 2014, Sanford developed and launched the Genetic Exploration of the Molecular Basis of Malignancy in Adults, or GEMMA, to determine if evaluating genetic information could help customize treatment options for adult patients whose cancer had progressed after the first line of treatment or was too rare for standard treatment. DNA was extracted from tumor samples and tested to identify targets for treatment.

Oncologist and cancer researcher Steven Powell, M.D., and his team used next-generation gene sequencing technology to analyze tumor samples for more than 100 patients. More than 90 percent of those patients had gene mutations that could impact their treatment, Powell reported. Some patients, for example, discovered they were eligible for a clinical trial or might benefit from other personalized medicine therapies. Nearly 40 percent of these patients were able to be treated with personalized therapies as a result of their testing. Many were treated on clinical trials with new drugs that previously would not have been available to them in this region.

“Molecular profiling programs like GEMMA don’t typically experience this degree of success,” said Powell. “Sixteen percent of our patients were able to go on clinical trials matching them to a personalized therapy; many academic centers are only able to do this five percent of the time. Our numbers indicate that the development of a molecular profiling program in a community setting in the Midwest is not only feasible but effective in getting patients access to the newest treatments.”

Enrollment concluded in late 2015, and results of GEMMA were outlined in an abstract published in conjunction with this year’s American Society of Clinical Oncology Annual Meeting held in Chicago last month. The published abstract can be found on the ASCO website.

Later this year, Sanford will begin the second version of GEMMA, which will integrate molecular profiling as part of standard cancer care. The study is called Community Oncology Use of Molecular Profiling to Personalize the Approach to Specialized Cancer Treatment at Sanford, or COMPASS. Sanford experts will analyze treatment plans based on molecular profiling to determine if outcomes improve. As part of GEMMA and COMPASS, the Sanford team has brought in more than 60 different personalized therapy options for patients through clinical trials in the past two years.

Spotting Dna Repair Genes Gone Awry

Researchers led by Ludwig Cancer Research scientist Richard Kolodner have developed a new technique for sussing out the genes responsible for helping repair DNA damage that, if left unchecked, can lead to certain cancers.

Genome instability suppressing (GIS) genes play an important role in correcting DNA damage involving the improper copying or reshuffling of large sections of chromosomes. Called gross chromosomal rearrangements, or GCRs, these structural errors can disrupt gene order or even result in an abnormal number of chromosomes.

“Mutated GIS genes have long been suspected of playing a role in the development of many types of cancers, but identifying them has been difficult due in large part to a lack of comprehensive GCR tests, or assays, in mammalian systems,” said Christopher Putnam, an associate investigator at the Ludwig Institute for Cancer Research, San Diego and one of the first authors of the study.

In the current issue of the journal Nature Communications, Putnam, Kolodner and their colleagues describe a novel two-pronged approach that combines methods from genetics and bioinformatics to identify GIS genes—first in yeast and then in humans.

“This is one of the first large-scale studies to integrate these two methods,” said co-senior author Sandro de Souza, a professor of Bioinformatics at the Federal University of Rio Grande do Norte’s Brain Institute in Brazil, who received Ludwig support for this study.

In the first step, the scientists used assays and technologies developed by Kolodner—who is director of the Ludwig Institute for Cancer Research, San Diego and a distinguished professor of Cellular and Molecular Medicine at the University of California, San Diego, School of Medicine—and his lab to screen thousands of mutant yeast strains for genes that suppress GCRs. They identified 182 GIS genes, 98 of which had not been described before. “Ours is probably one of the most comprehensive lists of GIS genes in yeast to date,” Putnam said.

The team also uncovered more than 400 previously unknown cooperating Genome Instability Suppressing genes (cGIS) genes, which only affect genome stability when combined with other mutations. “Before our experiment, only a few dozen cGIS genes were known. Now we know of hundreds,” said Putnam, who is also an adjunct assistant professor of Medicine at UC San Diego School of Medicine.

“These results have highlighted the complex genetic network that maintains genome integrity in normal cells,” said first author Anjana Srivatsan, a postdoctoral fellow in Kolodner’s lab at the Ludwig San Diego Branch and UC San Diego School of Medicine.

To determine how many of the yeast GIS genes had human counterparts implicated in cancers, the researchers searched The Cancer Genome Atlas (TCGA)—a compilation of genomic data from thousands of patients—for such human gene homologues.

They also supplemented their candidate list with human genes that are not found in yeast but that participate in the same pathways and protein complexes as the yeast GIS genes. “We didn’t want to just look for the human equivalent of yeast GIS genes because there are human GIS genes that don’t have yeast homologs,” Putnam said.

Three cancers in the TCGA were selected for screening: ovarian cancer, colorectal cancer and acute myeloid leukemia. The scientists hypothesized that a greater number of GIS gene defects should be implicated in ovarian cancer and colorectal cancer because these two cancers tend to involve numerous large-scale rearrangements of the genome. Leukemia served as an important control because it is a cancer with little genome instability, and thus should not involve any GIS gene defects.

As expected, the team found that 93% of ovarian cancers and 66% of colorectal cancers had genetic defects affecting one or more of the predicted GIS genes, whereas acute myeloid leukemia did not appear to have defects involving GIS genes.

The researchers are already screening more than a dozen other human cancers in the TCGA for GIS gene defects. “Understanding this process allows us to think more about how carcinogenesis proceeds and it might give us insights into defects that could be therapeutically actionable in the future,” said Putnam.

New Study Implicates Unusual Class of Circular RNAs in Cancer

Cancer cells are notorious for their genomes gone haywire, often yielding fusion proteins — mash-ups of two disparate genes that, once united, assume new and harmful capabilities. Exactly how such genome scrambling impacts RNA, particularly the vast and mysterious world of non-coding RNA, has been largely unexplored.

Now, a team led by investigators at Beth Israel Deaconess Medical Center (BIDMC) offers some early answers by studying an intriguing class of non-coding RNAs known as circular RNAs. Published in the March 31 advance online issue of Cell, their findings reveal that circular RNAs – like their protein counterparts – are also affected by genomic rearrangements in cancer, resulting in abnormal fusions. Moreover, these fusion-circular RNAs are not mere bystanders; they appear to promote tumor growth and progression, underscoring their role in the disease.

“Cancer is essentially a disease of mutated or broken genes, so that motivated us to examine whether circular RNAs, like proteins, can be affected by these chromosomal breaks,” said senior author Pier Paolo Pandolfi, MD, PhD, Director of the Cancer Center at BIDMC and George C. Reisman Professor of Medicine at Harvard Medical School. “Our work paves the way to discovering many more of these unusual RNAs and how they contribute to cancer, which could reveal new mechanisms and druggable pathways involved in tumor progression.”

When it comes to RNA, scientists’ worldview is in the midst of a significant shift. Long dismissed as a mere messenger, RNA is perhaps best known for its role ferrying instructions from the genome, which is cloistered in the nucleus, to more far-flung parts of the cell, where it is made into protein. Yet only 2 percent of the genome is copied (or “transcribed”) from DNA into RNA and then translated into protein. Scientists now recognize that much, if not all, of the remaining 98 percent — which had previously been deemed non-functioning— is in fact transcribed into RNA. The roles this vast swath of so-called “non-coding RNA” might play in human biology and disease now signify an area of intense research.

Curious about the possibility of circular RNAs contributing to cancer, Pandolfi and his colleagues set out to see if they could detect relevant changes in tumors known to harbor distinct fusion proteins, which result when different chromosomes abnormally join together, melding two separate genes into a new centaur-like gene. These chromosomal translocations are common in various types of leukemia, so the researchers examined two types: acute promyelocytic leukemia, which often carries a translocation between the PML and RARα genes; and acute myeloid leukemia, which can harbor a translocation between the MLL and AF9 genes.

The researchers found abnormal fusion-circular RNAs (f-circRNAs), corresponding to different exons associated with the PML-RARα gene fusion as well as the MLL-AF9 gene fusion. (Normally, multiple circular RNAs can be generated from a single gene, so it is not entirely surprising to find different f-circRNAs emerging from the same fusion gene.)

Remarkably, Pandolfi and his colleagues uncovered f-circRNAs in solid tumors, too — in samples from Ewing sarcoma, a form of soft tissue cancer, and lung cancer. Moreover, the team identified them using two distinct methods, PCR-based amplification as well as sequencing-based approaches, underscoring f-circRNAs as bona fide biological entities, rather than experimental artifacts.

“Our ability to readily detect these fusion-circular RNAs — and their normal, non-fused counterparts — will be enhanced by advances in sequencing technology and analytic methods,” said first author Jlenia Guarnerio, PhD, also of BIDMC. “Indeed, as we look ahead to cataloguing them comprehensively across all cancers and to deeply understanding their mechanisms of action, we will need to propel these new methodologies even further.”

To determine whether f-circRNAs play a functional role in cancer, the researchers introduced them experimentally into cells, causing the cells to increase their proliferation and tendency to overgrow — features shared by tumor cells. On the other hand, when the researchers blocked f-circRNA activity, the cells’ normal behaviors were restored.

The researchers also conducted experiments using a mouse model of leukemia. They focused on a specific f-circRNA associated with the MLL-AF9 fusion gene, called f-circM9. Although insufficient on its own to trigger leukemia, f-circM9 appears to work together with other cancer-promoting signals (such as the MLL-AF9 fusion protein) to cause disease. Additional studies suggest that f-circM9 may also help tumor cells persist in the face of anti-cancer drugs.

“These results are particularly exciting because they suggest that drugs directed at fusion-circular RNAs could be a powerful strategy to pursue for future therapeutic development in cancer,” said Pandolfi.

Circular RNAs were first identified more than three decades ago and largely dismissed as a rare cellular oddity. But a study published in 2012 by Patrick Brown’s group at Stanford University showed that they are present at high levels in diverse cell types, igniting scientists’ efforts to study and understand them. Surprisingly, circular RNAs — are among the most abundant non-coding RNAs in cells, driven in part by the molecules’ unusual chemical stability. Unlike linear RNAs, circular RNAs are not susceptible to RNA-degrading enzymes. This ability to persist makes them not only an interesting therapeutic target, but also a potential molecular beacon or biomarker that can facilitate the diagnosis of disease.

“Our knowledge of circular RNAs is really in its infancy,” explained Pandolfi. “We know that normally, they can bind proteins as well as DNA and microRNAs, but much more needs to be done to understand how fusion-circular RNAs work. We have only scratched the surface of these RNAs and their roles in cancer and other diseases.”

Study coauthors include BIDMC investigators Marco Bezzi, Jong Cheol Jeong, Stella V. Paffenholz, Kelsey Berry, Matteo M. Naldini, and Andrew H. Beck. Other coathors include Francesco Lo-Coco of the Università Tor Vergata in Italy, and Yvonne Tay of the National University of Singapore, in Singapore.