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Personal neoantigen vaccine prompts strong anti-tumor response in patients, study shows

A personal cancer treatment vaccine that targets distinctive “neoantigens” on tumor cells has been shown to stimulate a potent, safe, and highly specific immune anti-tumor response in melanoma patients, report scientists from Dana-Farber Cancer Institute and the Broad Institute of MIT and Harvard.

The study, published online by Nature “provides proof-of-principle that a personal vaccine tailored to a patient’s tumor can be produced and generates highly specific responses to that patient’s tumor after vaccination,” said the researchers, led by Catherine J. Wu, MD, senior author of the report. She is a researcher at Dana-Farber, the Broad Institute, and Harvard Medical School.

The scientists said that while most therapies are based on the on-size-fits-all model of medicine, “we’ve long recognized in cancer that every patient’s tumor is different. With recent advances in technology, it’s now becoming possible to create a therapy that’s suited to target an individual’s tumor.”

The researchers say the results warrant further development of neoantigen vaccines, both alone and in combination with other immunotherapy weapons such as checkpoint inhibitors. The vaccine, known as NeoVax, prompted strong activity by the patients’ immune systems while causing negligible side effects.

First authors of the report are Patrick A. Ott, MD, PhD, and Zhuting Hu, PhD, of Dana-Farber. Other senior authors include Nir Hacohen, PhD, of the Broad Institute and Massachusetts General Hospital, Edward Fritsch, PhD, formerly of Dana-Farber and now at Neon Therapeutics in Cambridge, Mass, and Eric Lander, PhD, of the Broad Institute.

Antigens are molecules that are displayed on the surface of cells and stimulate the immune system. Neoantigens are molecules on cell’s surfaces that are produced by DNA mutations that are present in cancer cells but not in normal cells, making neoantigens ideal targets for immune therapy against cancer, say the scientists. The vaccines used in the phase I trial contained up to 20 neoantigens, derived from an individual patient’s tumor. The vaccines were administered to patients to train their immune system to recognize these neoantigens, with the goal of stimulating the immune system to destroy the cancer cells that display them.

While other immunotherapies, such as checkpoint inhibitor drugs, also trigger immune responses against cancer neoantigens, they are not designed to be specific. They can also induce responses against normal tissue antigens, leading the immune system to attack normal tissues and cause toxicity in a subset of patients. The researchers found that the personal vaccine induced a focused T cell response against several tumor neoantigens, beyond what is normally seen in response to existing immunotherapies.

The vaccine was administered to six patients with melanoma whose tumors had been removed by surgery and who were considered at high risk for recurrence. The vaccinations were started at a median of 18 weeks after surgery. At a median of 25 months after vaccination, four of the six patients showed no evidence of cancer recurrence. In the other two patients, whose cancer had spread to their lungs, the disease recurred after vaccination. At that point, they began treatment with the drug pembrolizumab, which inhibits the PD-1 immune checkpoint. Both patients had complete resolution of their tumors and remain free of disease according to imaging scans.

The study results suggest, that a personalized neoantigen vaccine can potentially overcome two major hurdles in cancer therapy.

One is the heterogeneity of tumors – the fact that they are made up of cells with a variety of different traits, which often allows cancers to evade drugs targeted to malignant cells having a single genetic abnormality. The vaccine, because it contains many different neoantigens from the tumor, targets multiple genetic types of tumor cells. Wu added that in this respect, the response generated by a neoantigen vaccine is similar to the new wave of combination therapies, which are showing more promise in treating cancers that typically develop resistance to single drugs. “We are leveraging the immune system’s natural ability to detect and attack many target antigens, as it does every time we get an infection,” she said.

A second hurdle in cancer is to generate an immune response sharply focused on cancer cells while avoiding normal cells and tissues. This aim was achieved by the vaccine, which appeared to have few “off-target” effects, causing only flu-like symptoms, fatigue, rashes, and irritation at the site of the vaccine injection, according to the report.

Despite decades of attempts to develop effective cancer treatment vaccines, they have mostly failed at producing potent antitumor immune responses. The study authors say that is because these vaccines have generally been made with tumor antigens that are too similar to antigens on normal cells: as a result, the body generates a weaker immune response to avoid harming normal cells, a process called immune tolerance. By contrast, the neoantigen vaccine is custom-made for each patient using antigens produced by mutations unique to the patient’s cancer and only present on cancer cells, thus bypassing the nature immune tolerance process.

To create the vaccine, samples of a patient’s tumor and normal DNA from the patient’s blood underwent whole-exome sequencing to reveal mutations present only in the tumor’s genetic program. Because some mutations are present in the DNA but the gene is not made into RNA and protein, the researchers used RNA sequencing to identify mutations that caused the production of a mutated RNA, which is then normally translated into a protein.

Since T cells can only recognize neoantigens that are “presented” to them by HLA molecules of the immune system, a key step in making the vaccine is using computer algorithms to predict which neoantigen peptides will bind strongly to the HLA molecules for recognition by T cells. Algorithms, such as NetMHC, have been developed in recent years, making it feasible to select HLA-binding neoantigen peptides for the vaccine. Applying this tool to the six patients’ tumor samples yielded dozens of unique neoantigens for each patient’s personal vaccine.

Finally, the selected neoantigen peptides were synthesized and mixed with an adjuvant – a biochemical substance that helps to jump-start the immune response. The vaccine was then injected under the skin of the patient, with five priming doses followed by two booster doses of the vaccine.

The vaccine was aimed at generating responses to the neoantigens from T cells of two kinds – CD8+ killer cells and CD4+ helper cells. When the team monitored the vaccine’s effects on the immune system in each patient, they found that both T cell types had indeed been activated by the vaccine and could recognize the neoantigens bound to HLA molecules. Most importantly, many of the T cells were able to recognize the tumor cells directly, demonstrating that the vaccine had triggered a tumor-specific immune response that could target the patient’s tumor.

“Future neoantigen vaccine trials will recruit more patients with advanced disease to test the efficacy of the vaccine, take advantage of improved methods for predicting antigen presentation to boost the number of effective neoantigens and test for synergy with checkpoint blockade and other immunotherapeutics,” the scientists said. “If successful in subsequent trials, a personal vaccine has the potential to be applied to any cancer that harbors a sufficient numbers of neoantigens for vaccination.”

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First Large-Scale Genomic Analysis of Key Acute Leukemia Will Likely Yield New Therapies

A consortium including St. Jude Children’s Research Hospital and the Children’s Oncology Group has performed an unprecedented genomic sequencing analysis of hundreds of patients with T-lineage acute lymphoblastic leukemia (T-ALL). The results provide a detailed genomic landscape that will inform treatment strategies and aid efforts to develop drugs to target newly discovered mutations.

The data will also enable researchers to engineer better mouse models to probe the leukemia’s aberrant biological machinery. The study was a collaboration between the St. Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project, the Children’s Oncology Group (COG) and the National Cancer Institute’s Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative

The project’s 39 researchers were led by Charles Mullighan, M.D., MBBS, a member of the St. Jude Department of Pathology, with co-corresponding authors Jinghui Zhang, Ph.D., chair of the St. Jude Department of Computational Biology and Stephen Hunger, M.D., of the Children’s Hospital of Philadelphia. The research was selected for advance online publication today in the journal Nature Genetics.

“This first comprehensive and systematic analysis in a large group of patients revealed many new mutations that are biologically significant as well as new drug targets that could be clinically important,” Mullighan said. “Leukemias typically arise from multiple genetic changes that work together. Most previous studies have not had the breadth of genomic data in enough patients to identify the constellations of mutations and recognize their associations.”

T-ALL is a form of leukemia in which the immune system’s T cells acquire multiple mutations that freeze the cells in an immature stage, causing them to accumulate in the body. ALL is the most common type of childhood cancer, affecting about 3,000 children nationwide each year. T-ALL constitutes about 15 percent of those cases. While about 90 percent of children with ALL can be cured, many still relapse and require additional treatment.

The multi-institutional effort involved sequencing the genomes of 264 children and young adults with T-ALL—the largest such group ever analyzed. The study involved sophisticated analysis of multiple types of genomic data, led by Yu Liu, Ph.D., a postdoctoral fellow in Zhang’s Computational Biology laboratory and first author of the study. Their analyses identified 106 driver genes—those whose mutations trigger the malfunctions that block normal T cell development and give rise to cancer. Half of those mutated genes had not been previously identified in childhood T-ALL.

The study enabled the researchers to compare the frequencies of mutations among patients whose cancerous cells were sequenced at the same detailed level, Mullighan said. Also important, he said, was that all the patients had uniform treatment, which enabled the researchers to draw meaningful associations between the genetics of their cancer and the response to different treatments. Such associations will enable better diagnosis and treatment of T-ALL with existing drugs.

Researchers analyzed the cancerous T cells as well as those that treatments had rendered non-cancerous. Comparing the two populations of cells could reveal valuable clues about why specific treatments were successful in thwarting particular cancer-causing mutations.

The findings revealed significant unexpected findings. “We went into this study knowing that we didn’t know the full genomic landscape of T-ALL,” Hunger said. “But we were surprised that over half of the new targets and mutations were previously unrecognized. It was particularly unexpected and very striking that some mutations were exclusively found in some subtypes of T-ALL, but not others.”

Cancers are driven by mutations in genes that are the blueprint for protein enzymes in signaling pathways in cells—the biological equivalent of circuits in a computer. While a cancer may arise from an initial founding mutation, that mutation triggers a cascade of other mutations that help drive the cancer.

The new genomic analysis confirmed that T-ALL was driven by mutations in known signaling pathways, including JAK–STAT, Ras and PTEN–PI3K.

However, the new analysis identified many more genetic mutations in those known pathways. The findings offered more targets for drugs to shut down the aberrant cells. “So the frequency of the patients that are potentially amenable to these targeted approaches is higher than we appreciated before,” Mullighan said.

The researchers also found cases in which the same T-ALL subtype had mutations in different pathways triggered by the same cancer-causing founding mutation. “We believe this finding suggests we can target such subtypes with an inhibitor drug for one of the pathways, and it’s likely to be effective,” Mullighan said.

The multitude of new mutations uncovered in the new study will also enable researchers to use genetic engineering to create mouse models that more accurately reflect human cancer, he said. Such models are invaluable for understanding the biological machinery of T-ALL, as well as testing new drug strategies. “We now have a launching pad, if you will, to design mouse models that include multiple genetic mutations to more faithfully reflect the leukemias we see in humans,” Mullighan said.

The research also offers a broader lesson for genomic studies of cancers, Zhang said. “Our study is further evidence that if you systematically study a large enough population with careful, detailed genomic analysis, you will discover new mutational patterns of collaboration or exclusion across multiple genes unique to each T-ALL subtype,” she said.

. COG is a federally supported clinical trials group focused exclusively on childhood cancer. TARGET uses genomic analysis of COG samples to identify therapeutic targets and spur development of more effective treatment for childhood cancer.

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Heart disease, leukemia linked to dysfunction in nucleus

We put things into a container to keep them organized and safe. In cells, the nucleus has a similar role: keeping DNA protected and intact within an enveloping membrane. But a new study by Salk Institute scientists, detailed in the November 2 issue of Genes & Development, reveals that this cellular container acts on its contents to influence gene expression.

“Our research shows that, far from being a passive enclosure as many biologists have thought, the nuclear membrane is an active regulatory structure,” says Salk Professor Martin Hetzer, who is also holder of the Jesse and Caryl Philips Foundation chair. “Not only does it interact with portions of the genome to drive gene expression, but it can also contribute to disease processes when components are faulty.”

Using a suite of molecular biology technologies, the Salk team discovered that two proteins, which sit in the nuclear envelope, together with the membrane-spanning complexes they form, actively associate with stretches of DNA to trigger expression of key genes. Better understanding these higher-level functions could provide insight into diseases that appear to be related to dysfunctional nuclear membrane components, such as leukemia, heart disease and aging disorders.

Historically, the nuclear membrane’s main purpose was thought to be keeping the contents of the nucleus physically separated from the rest of the cell. Complexes of at least thirty different proteins, called nucleoporins, form gateways (pores) in the membrane, controlling what goes in or out. But as the Hetzer lab’s work on nucleoporins shows, these nuclear pore complexes (NPCs), beyond being mere gateways into the nucleus, have surprising regulatory effects on the DNA inside.

“Discovering that key regulatory regions of the genome are actually positioned at nuclear pores was very unexpected,” says Arkaitz Ibarra, a Salk staff scientist and first author of the paper. “And even more importantly, nuclear pore proteins are critical for the function of those genomic sites.”

Curious about all the regions of DNA with which nucleoporins potentially interact, the team turned to a human bone cancer cell line. The scientists used a molecular biology technique called DamID to pinpoint where two nucleoporins, Nup153 and Nup93, came into contact with the genome. Then they used several other sequencing techniques to understand which genes were being affected in those regions, and how.

The Salk team discovered that Nup153 and Nup93 interacted with stretches of the genome called super-enhancers, which are known to help determine cell identity. Since every cell in our body has the same DNA, what makes a muscle cell different from a liver cell or a nerve cell is which particular genes are turned on, or expressed, within that cell. In the Salk study, the presence of Nup153 and Nup93 was found to regulate expression of super-enhancer driven genes and experiments that silenced either protein resulted in abnormal gene expression from these regions. Further experiments in a lung cancer cell line validated the bone cancer line results: Nucleoporins in the NPC were found to interact with multiple super-enhancer regions to drive gene expression, while experiments that altered the NPC proteins made related gene expression faulty, even though the proteins still performed their primary role as gatekeepers in the cell membrane.

“It was incredible to find that we could perturb the proteins without affecting their gateway role, but still have nearby gene expression go awry,” says Ibarra.

The results bolster other work indicating that problems with the nuclear membrane play a role in heart disease, leukemia and progeria, a rare premature aging syndrome.

“People have thought the nuclear membrane is just a protective barrier, which is maybe the reason why it evolved in the first place. But there are many more regulatory levels that we don’t understand. And it’s such an important area because so far, every membrane protein that has been studied and found to be mutated or mis-localized, seems to cause a human disease,” says Hetzer.

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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.

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New Treatment Leaves Liver Cancer Cells In Limbo

Scientists have shown that a mutation in a gene called Arid1b can cause liver cancer. The gene normally protects against cancer by limiting cell growth, but when mutated it allows cells to grow uncontrollably. The researchers have shown that two existing drugs can halt this growth in human cells. This points to a new approach to treating liver cancer.

These early results could be translated into a treatment relatively quickly, says Jesus Gil of the MRC Clinical Sciences Centre (CSC), based at Imperial College London, and who led the study. This is because the drugs are already used to treat other types of cancer. Gemcitabine is used on bladder, pancreas and ovary cancer, whilst DON has been tested in clinical trials. Both are known to be safe in people so will not require the usual toxicity testing.

According to MacMillian Cancer Support, 4200 people in the UK are diagnosed with liver cancer annually. “Liver cancer is a deadly disease,” says Luca Tordella, a postdoctoral researcher at the CSC who played a key role in the study. He says the new treatment will be most effective in people who have a mutation in the Arid1b gene. “It’s important to better classify patients into groups, according to their genes. One advance of personalised medicine is to understand which drug will work best on you, and which on me.”

The CSC team began by looking for mutations in the DNA of 100 people with liver cancer. They focused in on genes known to regulate a cellular state called senescence. This is a built-in safety mechanism that helps to protect the cell against cancer. Senescence is triggered when a cell grows abnormally fast and, once induced, it acts to keep the cell in limbo, such that it is alive but can no longer grow or divide. Bypassing senescence is a hallmark of many types of cancer.

It has previously been suggested that the Arid1b gene plays a role in liver cancer. The CSC team are the first to show that it does so by disrupting senescence. Working with researchers at the University of Tübingen, Germany, they mutated Arid1b in mice and human cells, and this stopped the cells from entering senescence. When Arid1b was mutated in mice that also had a mutation in the gene Ras, the mice developed liver cancer.

The researchers also identified exactly how Arid1b stimulates senescence. In healthy cells, Arid1b is part of a bigger complex (called SWI/SNF) that regulates the activity of hundreds of genes. One of these genes produces an enzyme that breaks down the building blocks of DNA. Without these blocks, the cell cannot continue to grow and divide so enters senescence. But if Arid1b is mutated, the blocks cannot be degraded and the cell continues to grow, bypassing senescence and potentially growing out of control.

Gil and Tordella have shown that it’s possible to induce senescence by treating human cells, which have an Arid1b mutation, with either gemcitabine or DON. Both drugs inhibit the synthesis of these blocks, called nucleotides, and thereby induce senescence and stop cancer in its tracks.

“Around 20% of patients with liver cancer have mutations on the genes encoding for components of the SWI/SNF complex. What we suggest is if we treat these people with drugs that target the degradation of nucleotides, they will respond,” says Gil. “We plan to continue to research this in the lab to develop treatments to target liver cancer.”

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Cancer Sequencing Results Differ Based On Genetic Background Of Comparison Genome

When University of Colorado Cancer Center researcher, Jing Hong Wang, MD, PhD, found more than 1,000 genetic translocations in her mouse model of B cell lymphoma, she assumed her lab had made a mistake. To rule out experimental technique as the cause of the way-more-than-expected genomic alterations, Wang’s lab sequenced three different types of cells from “wildtype” mice – effectively the kind that might move into your garage in bad weather. Like the lymphoma cells before them, the cells from wildtype mice also had over a 1,000 translocations.

“We thought ‘let’s just do another practice’,” says Wang, also an associate professor in the CU School of Medicine Department of Immunology & Microbiology.

For “practice”, paper co-first author, Katherine Gowan, downloaded new mouse genomic data from the website of Wellcome Trust Sanger Institute outside Cambridge in the UK, one of the world’s leading institutes for genetic research. Gowan is a researcher with the group of Kenneth Jones, PhD, co-director of the CU Cancer Center Bioinformatics Shared Resource.

“When we mapped the genome of this particular mouse strain against the mouse reference genome published by the National Center for Biotechnology Information, we found thousands of translocations, even more than our experimental model!” Wang says.

The problem was not their experimental mouse. The problem was not the quality of their data nor the computational algorithm they used to discover translocations. The problem, as reported in an article in the journal BMC Genomics, was that reference genomes are different for various mouse strains. Not all mice have the same DNA sequences in the same locations on their chromosomes – due to this genetic variation, the DNA sequences of one mouse strain may appear out of place when compared with the DNA sequences of any other mouse strain.

The goal of this research was to discover new translocations that could be driving lymphoma. These translocations – accidental genetic rearrangements in which a gene is snipped from one location and pasted into another, sometimes creating a “fusion gene” made from both – have been implicated in a range of cancers, for example ALK-positive lung cancer, which is driven by the translocation of the ALK gene, which fuses with the gene EML4. The question was whether a similar translocation might be to blame for a subset of lymphomas.

“Unfortunately, when we have so many events, the artifacts may mask our real events,” says Wang, meaning that with thousands of translocations identified by next-generation sequencing, it was almost impossible to discover the “needle” of a potentially oncogenic translocation amid the “haystack” of identified translocations that were, in fact, only the unimportant, random differences between individual mouse genomes.

“Then we started to think about all these human cancer genomic studies,” Wang says. “People use all this sequencing data to show genomic changes in human cancers, but what if these studies have similar comparison problems?”

First, Wang points out, this possible trap is irrelevant when analyzing a patient’s cancer for any known genetic change. In the previous example of lung cancer, genomic testing (often using the technique of fluorescent in situ hybridization or FISH) can tell if a cell’s chromosomes do or do not contain an ALK-EML4 fusion gene. But it is when searching for important differences between a human cancer cell and a healthy human cell that the genetic backgrounds of these cells may skew results – due to the randomness of repeats and gene polymorphisms and other unpredictable genetic variations, the differences between a cancerous and a healthy cell may be due to chance and not to the influence of the cancer at all.

Part of the problem is the small size of genetic “snips” used by today’s next-generation sequencing technology. In “next-gen seq” the machine reads a test genome as many snips, each made up of 100 to 150 base pairs. Then the computational biologist fits these snips like puzzle pieces against a reference genome. When there is a match, the system puts the piece in place and thus, because it knows the makeup of the reference genome, can come to know the makeup of the test genome. Unfortunately, with 3 billion base pairs in the human genome, there may be many false matches for short, 100 base-pair snips. Technology is on the way to solve this problem, sequencing the genome in much longer snips (1,000 or more base pairs).

Until then, Wang suggests a possible fix: “We suggest considering not mapping your data to a reference genome, but to the genome of some cell from the same source that doesn’t have cancer.”

The paper calls this process “de novo assembly” – basically, instead of comparing a cancerous apple to a healthy orange, it is comparing a cancerous apple to a healthy apple.

“People should be their own control. Instead of working with the published, generic reference genome, we should work with two samples (control vs. cancer) from the same person,” Wang says. “Only then can you really figure out what’s going on in your cancer cell genome.”

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Asthma Research Unexpectedly Yields New Treatment Approach For Inherited Enzyme Disease

Experiments designed to reveal how a protein protects the lungs from asthma-related damage suggest a new way to treat a rare disease marked by the inability of cells to break down fats, according to a report in EBioMedicine published online Oct. 25.

The study results address Gaucher’s disease, which is caused by a genetic glitch in cell structures called lysosomes that process fats and remove cellular waste. Found mostly in Jews of Eastern and Central European origin, the condition may come with joint pain, blood disorders, enlarged spleens and livers, memory loss, and lung damage.

At a cellular level, Gaucher’s disease is associated with abnormally low production of the protein progranulin, as well as with the misplaced buildup of the enzyme beta-glucocerebrosidase, or GBA, outside lysosomes, instead of inside where it is needed.

Led by researchers from NYU Langone Medical Center, the new study found that a manufactured version of progranulin reversed most effects of Gaucher’s disease in mouse and human cell studies, including GBA accumulation.

“Our results suggest a new way to treat Gaucher’s disease that corrects abnormal enzyme delivery by progranulin to lysosomes, as opposed to current treatment strategies that temporarily replenish lysosomal GBA stores, which are then steadily consumed,” says senior study investigator Chuanju Liu, PhD, a professor in the Departments of Orthopaedic Surgery and Cell Biology at NYU Langone. The research team and NYU Langone hold a patent on related, potential therapies.

Among the study’s other key findings was that progranulin must bind to other molecules to transport the enzyme to lysosomes, specifically the protective “heat shock” protein 70. If unshielded, cellular GBA molecules fold up and stick together outside lysosomes.

Researchers also found that adding synthetic progranulin, or Pcgin, to blood cells obtained from patients with Gaucher’s, led to a 40 percent reduction in GBA clumping within a week. Pcgin was used because it is chemically more stable than progranulin and poses no risk of uncontrolled tumor-like cell growth in test animals, say the authors.

“Our new experiments are the first to explain why reduced progranulin is a key characteristic of Gaucher’s, and why the mice engineered to lack the protein serve as such a good model to test new therapies,” says lead study investigator Jian Jinlong, MD, PhD, an associate research scientist at NYU Langone.

Along with their role in brain disorders, progranulin shortages had been tied by previous research to cell swelling in asthmatic lungs. In the current set of experiments in progranulin-deficient mice, adding Pcgin reduced lung-tissue swelling by as much as 60 percent, an effect seen with current GBA-replacement treatments.

According to Liu, further research is needed to determine the precise mechanism by which progranulin reduces cell swelling, a process that would likely yield even more drug targets for Gaucher’s disease.

Experts estimate that as many as one in 50,000 Americans has some form of Gaucher’s, while one in 500 Jews of Ashkenazi descent has the disease.

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Cytomegalovirus Infection Relies On Human RNA-Binding Protein

Viruses hijack the molecular machinery in human cells to survive and replicate, often damaging those host cells in the process. Researchers at the University of California San Diego School of Medicine discovered that, for cytomegalovirus (CMV), this process relies on a human protein called CPEB1. The study, published October 24 inNature Structural and Molecular Biology, provides a potential new target for the development of CMV therapies.

“We found that CPEB1, one of a family of hundreds of RNA-binding proteins in the human genome, is important for establishing productive cytomegalovirus infections,” said senior author Gene Yeo, PhD, professor of cellular and molecular medicine at UC San Diego School of Medicine.

CMV is a virus that infects more than half of all adults by age 40, and stays for life. Most infected people are not aware that they have CMV because it rarely causes symptoms. However, CMV can cause serious health problems for people with compromised immune systems, or babies infected with the virus before birth. There are currently no treatments or vaccines for CMV.

In human cells, RNA is the genetic material that carries instructions from the DNA in a cell’s nucleus out to the cytoplasm, where molecular machinery uses those instructions to build proteins. CPEB1 is a human protein that normally binds RNAs that are destined to be translated into proteins.

Yeo’s team discovered that CPEB1 levels increase dramatically in human cells infected by CMV. Using genomics technologies, the researchers also found that increased CPEB1 levels in CMV-infected cells leads to abnormal processing of RNAs encoding thousands of human genes. In addition, they were surprised to find that CPEB1 was necessary for proper processing of viral RNAs. Without the host CPEB1 protein, viral RNA did not mature properly and the virus was weakened.

CMV-infected human cells undergo abnormal changes and produce more virus, which ultimately infects other cells. In collaboration with Deborah Spector, PhD, Distinguished Professor at UC San Diego School of Medicine and Skaggs School of Pharmacy and Pharmaceutical Sciences, the team went on to show that suppressing CPEB1 levels during CMV infection reversed these harmful cellular changes and reduced viral production tenfold.

“CPEB1 was previously shown to play a role in neuronal development and function, but this involvement in active viral infections is unexpected,” said first author Ranjan Batra, PhD, a postdoctoral fellow in Yeo’s lab. “This discovery has important implications for many viral infections.”

Yeo said the next steps are to determine the therapeutic value of inhibiting CPEB1 in CMV infections and identify other RNA-binding proteins that may be important in other viral infections.

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Genome Engineering Paves The Way For Sickle Cell Cure

A team of physicians and laboratory scientists has taken a key step toward a cure for sickle cell disease, using CRISPR-Cas9 gene editing to fix the mutated gene responsible for the disease in stem cells from the blood of affected patients.

For the first time, they have corrected the mutation in a proportion of stem cells that is high enough to produce a substantial benefit in sickle cell patients.

The researchers from the University of California, Berkeley, UCSF Benioff Children’s Hospital Oakland Research Institute (CHORI) and the University of Utah School of Medicine hope to re-infuse patients with the edited stem cells and alleviate symptoms of the disease, which primarily afflicts those of African descent and leads to anemia, painful blood blockages and early death.

“We’re very excited about the promise of this technology,” said Jacob Corn, a senior author on the study and scientific director of the Innovative Genomics Initiative at UC Berkeley. “There is still a lot of work to be done before this approach might be used in the clinic, but we’re hopeful that it will pave the way for new kinds of treatment for patients with sickle cell disease.”

In tests in mice, the genetically engineered stem cells stuck around for at least four months after transplantation, an important benchmark to ensure that any potential therapy would be lasting.

“This is an important advance because for the first time we show a level of correction in stem cells that should be sufficient for a clinical benefit in persons with sickle cell anemia,” said co-author Mark Walters, a pediatric hematologist and oncologist and director of UCSF Benioff Children’s Hospital Oakland’s Blood and Marrow Transplantation Program.

The results will be reported in the Oct. 12 issue of the online journal Science Translational Medicine.

Sickle cell disease is a recessive genetic disorder caused by a single mutation in both copies of a gene coding for beta-globin, a protein that forms part of the oxygen-carrying molecule hemoglobin. This homozygous defect causes hemoglobin molecules to stick together, deforming red blood cells into a characteristic “sickle” shape. These misshapen cells get stuck in blood vessels, causing blockages, anemia, pain, organ failure and significantly shortened lifespan. Sickle cell disease is particularly prevalent in African Americans and the sub-Saharan African population, affecting hundreds of thousands of people worldwide.

The goal of the multi-institutional team is to develop genome engineering-based methods for correcting the disease-causing mutation in each patient’s own stem cells to ensure that new red blood cells are healthy.

The team used CRISPR-Cas9 to correct the disease-causing mutation in hematopoietic stem cells – precursor cells that mature into red blood cells – isolated from whole blood of sickle cell patients. The corrected cells produced healthy hemoglobin, which mutated cells do not make at all.

Future pre-clinical work will require additional optimization, large-scale mouse studies and rigorous safety analysis, the researchers emphasize. Corn and his lab have joined with Walters, an expert in developing curative treatments such as bone marrow transplant and gene therapy for sickle cell disease, to initiate an early-phase clinical trial to test this new treatment within the next five years.

Notably, research groups might be able to apply the approach described in this study to develop treatments for other blood diseases such as β-thalassemia, severe combined immunodeficiency (SCID), chronic granulomatous disease, rare disorders like Wiskott-Aldrich syndrome and Fanconi anemia, and even HIV infection.

“Sickle cell disease is just one of many blood disorders caused by a single mutation in the genome,” Corn said. “It’s very possible that other researchers and clinicians could use this type of gene editing to explore ways to cure a large number of diseases.”

“There is a clear path for developing therapies for certain diseases,” said co-senior author Dana Carroll of the University of Utah, who co-developed one of the first genome editing techniques over a decade ago. “It’s very gratifying to see gene editing technology being brought to practical applications.”

The work is the fruit of the Innovative Genomics Initiative, a joint effort between UC Berkeley and UCSF that aims to correct DNA mutations that underlie human disease using CRISPR-Cas9, a pioneering technology co-developed by scientists at UC Berkeley that has made genome editing easier and more efficient than ever before.

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New Non-Invasive Assay May Improve Surveillance Of Heart And Other Solid-Organ Transplants

Patients who have received a solid organ transplant require lifelong immunosuppressive therapy. The threat of transplant rejection due to insufficient drug therapy must be balanced against increased risks of infections and cancer from excessive immunosuppression. A significant unmet need exists for non-invasive diagnostic tools to monitor transplant recipients, especially for early detection of active injury and rejection. A report in The Journal of Molecular Diagnostics describes a new non-invasive test that measures donor-derived cell-free DNA (dd-cfDNA) in plasma that has the potential to reduce complications and rejection, improving outcomes in transplant recipients.

“dd-cfDNA is an emerging biomarker of transplanted organ injury, and the availability of a clinical-grade, analytically validated assay is critical for advancement of this biomarker toward improving the outcomes of transplant patients,” explained lead investigator Marica Grskovic, PhD, Associate Director, R&D, CareDx, Inc. (Brisbane, CA).

Plasma cfDNA has been proposed as a biomarker for prenatal testing, cancer, and organ transplantation. Taking advantage of genetic differences between a transplant donor and recipient, techniques have been developed to measure levels of a donor’s DNA in the recipient’s plasma, serum, or urine as a way to monitor the health of transplanted tissue, whether from the heart, lungs, liver, or other organs.

Although dd-cfDNA assays for research have been described previously, this is the first time a clinical-grade assay has been reported. The new assay detects plasma dd-cfDNA within the range of levels evident from transplant patient samples.

An advantage of the new next-generation sequencing (NGS)-based amplification assay is that it does not require determination of the donor’s and recipient’s genotype, a process which requires significant time, cost, and tissue availability. Although tissue biopsy is another way to monitor a transplanted organ, it is invasive, time consuming, costly, and risky. The new assay can be completed within three days, which can be important for clinical decision-making.

In the current report, data are presented from a multi-center heart transplantation study showing that dd-cfDNA was, on average, three-fold higher in patients experiencing acute rejection than in stable transplant recipients without acute rejection. A decrease in dd-cfDNA levels upon successful anti-rejection treatment was also observed.

Hannah Valantine, MD, Senior Investigator NHLBI, and NIH Chief Officer for Scientific Workforce Diversity, stated, “In collaboration with colleagues Drs. Stephen Quake, Kiran Khush, and Iwijn De Vlaminck at Stanford, we performed the pioneering research studies using NGS for heart and lung transplant. I am delighted to see this technology translating into a clinical-grade assay to which patients will have access to improve the precision of patient management.”

The researchers expect the assay to be useful for monitoring other types of transplanted organs. Additional multi-centered observational studies for heart and kidney transplant patients are underway to further evaluate the assay’s clinical validity and utility. The assay is currently validated only for single organ donor/recipient pairs.

“These results show promise in using cfDNA not only to detect rejection, but also to monitor response to treatment. The ongoing measurement of cfDNA may allow clinicians to better personalize care, adjust immunosuppression regimens, and improve the long-term outcomes of transplant recipients,” noted Dr. Grskovic.

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For Normal Heart Function, Look Beyond The Genes

Researchers have shown that when parts of a genome known as enhancers are missing, the heart works abnormally, a finding that bolsters the importance of DNA segments once considered “junk” because they do not code for specific proteins.

The team, led by scientists at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab), examined the role of two heart enhancers in the mouse genome, showing that the loss of either one resulted in symptoms that resemble human cardiomyopathy, a disease in which the heart muscle often becomes enlarged or rigid. In humans, the disease often leads to heart failure.

The findings appear in a study to be published Oct. 5 in the journal Nature Communications.

In that same paper, the researchers provided a comprehensive genome-wide map of more than 80,000 enhancers considered relevant to human heart development and function. The two heart enhancers that they tested were the mouse equivalent of enhancers chosen from among that catalog.

“The cardiac changes that we observed in knockout mice lacking these enhancers highlight the role of noncoding sequences in processes that are important in human disease,” said study co-senior author Axel Visel, senior staff scientist and one of three lead researchers at the Mammalian Functional Genomics Laboratory, part of Berkeley Lab’s Environmental Genomics and Systems Biology (EGSB) Division. “Identifying and interpreting sequence changes affecting noncoding sequences is increasingly a challenge in human genetics. The genome-wide catalog of heart enhancers provided through this study will facilitate the interpretation of human genetic data sets.”

Study lead author Diane Dickel, project scientist, and co-senior author Len Pennacchio, senior staff scientist, both work with Visel at Berkeley Lab’s Mammalian Functional Genomics Laboratory.

DNA Dark Matter

When scientists sequenced the human genome, they discovered that less than 5 percent of our DNA were genes that actually coded for protein sequences. The biological functions of the noncoding portions of the genome were unclear.

Over the past fifteen years, however, there has been a growing appreciation for the importance of these noncoding regions, thanks in large part to the efforts of individual labs and, more recently, large international efforts such as the Encyclopedia of DNA Elements (ENCODE) project.

What became clear from this work is that there are many elements of the genome, including enhancers, that are involved in regulating gene expression, even though they do not encode for proteins directly.

This realization meant that there were vast sections of the genome that needed to be explored and understood. Dickel noted that there are about 20,000 genes in the mouse genome, and in many cases, scientists have a fairly good understanding of what will happen if any one of them is disabled. In contrast, there are 80,000 candidate heart enhancers in the human genome, and it is still unclear how important they are for human development.

“In genetic studies, the way you establish whether a gene is important is you delete it from the genome and see what happens,” said Dickel. “In many cases, there are genes that, if disabled, make it difficult for the organism to survive. For enhancers, it’s less known what the consequences are if they are damaged or missing. To use a car analogy, if we took the battery out of a car, it wouldn’t start. That’s a critical component. A missing or damaged enhancer could be essential like a battery, or more similar to a missing passenger seat in the car. It’s not as nice, but it’s still possible to drive the car.”

Mapping and Testing the Enhancers

To assess the function of heart enhancers, the researchers first compiled a single road map to guide them. They used results from a technology called ChIP-seq (chromatin immunoprecipitation sequencing) to identify the likely heart enhancers in the human genome.

The researchers say this map will become an important tool as advances in genomics usher in a new era of personalized medicine.

“This compendium of human heart enhancers will be a valuable resource for many disease researchers who have begun adopting whole genome sequencing of patients to look for disease-causing mutations in both the coding and noncoding portion of the genome,” said Dickel.

Using the map, the researchers picked two enhancers located near genes associated with human heart disease. They then determined their equivalent enhancers on the mouse genome and disabled them in mice.

They compared the mice with the disabled enhancers with control mice that had no mutation and saw very large changes in gene expression in the test mice.

Echocardiograms used to image the hearts from the two groups of mice confirmed that the heart tissue of mice with a disabled enhancer was pumping with less power than normal, consistent with the signs of human cardiomyopathy.

“Prior to this work, no study had looked at what happens to heart function as a result of knocking out the heart enhancers in the genome,” said Dickel. “What was surprising to me was that outwardly, the knockout mice seemed fine. If you just looked at them, you wouldn’t necessarily see anything wrong.”

With so many enhancers to test, the map could help scientists prioritize which ones to assess in animal studies and in disease research, the researchers said.

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Researchers Find Fertility Genes Required For Sperm Stem Cells

The underlying cause of male infertility is unknown for 30 percent of cases. In a pair of new studies, researchers at University of California San Diego School of Medicine determined that the reproductive homeobox (RHOX) family of transcription factors — regulatory proteins that activate some genes and inactivate others — drive the development of stem cells in the testes in mice. The investigators also linked RHOX gene mutations to male infertility in humans. The mouse study is published September 27 byCell Reports and the human study was published September 15 by Human Molecular Genetics.

“Infertility in general, and especially male fertility, gets little attention considering how common of a problem it is — about 15 percent of couples are affected, and nearly half of these cases are due to male infertility,” said Miles Wilkinson, PhD, professor of reproductive medicine at UC San Diego School of Medicine and senior author of the Cell Reports study. “That means around 7 percent of all males of reproductive age — nearly 4 million men in the U.S. — have fertility problems.” Wilkinson is also a co-author of theHuman Molecular Genetics study, which was led by Jörg Gromoll, PhD, at the University of Münster in Germany.

Sperm are made from cells that undergo many stages. Transcription factors have been identified that direct most of these cell stages, from the dividing cells in the embryo to the cells that rearrange and partition the chromosomes to individual “pre-sperm” in the testes. However, before this latest research, Wilkinson said no transcription factors were known to direct one of the most critical stages — the formation of the stem cells in the testes, known as spermatogonial stem cells.

In the Cell Reports study, Wilkinson and team removed the entire cluster of 33 Rhox genes in mice. They were surprised to find that the most notable defect in these mice was a deficiency in spermatogonial stem cells. Hye-Won Song, PhD, assistant project scientist in Wilkinson’s lab and first author of the Cell Reports study, removed just one of the Rhoxgenes — Rhox10 – and found essentially the same defect as deleting the full set.

Wilkinson, Song and team discovered there was nothing wrong with the spermatogonial stem cells in mice lacking Rhox10, only that there were so few. They found that this occurred because most of the earlier stage cells — pro-spermatogonia — did not specialize into spermatogonial stem cells. As a result, the testes of Rhox10-deficient mice did not enlarge and their sperm counts failed to increase as they aged.

The researchers concluded that Rhox10 is the most critical gene in the Rhox cluster, and that it plays a role in spermatogonial stem cell formation.

The Rhox genes are on the X chromosome. It makes sense that male infertility would be caused by mutated genes on the X chromosome, Wilkinson said, because men only have one copy — if something goes wrong with an X-linked gene, they don’t have a backup, like women do.

There are several potential clinical implications of these results, the researchers said. For example, Rhox genes may have roles in testicular tumors that arise from germ cells that failed to convert into spermatogonial stem cells and thus are “frozen” at the pro-spermatogonia stage. Rhox genes may also be useful for regenerative medicine approaches to restoring fertility through therapy with spermatogonial stem cells.

In the second study, published by Human Molecular Genetics, Gromoll and colleagues sequenced RHOX genes in 250 men with severely low sperm count. They found two mutations in one of these genes (RHOXF1) and four mutations in the other two (RHOXF2and RHOXF2B, which are almost identical). Only one mutation was also found in a control group of men with normal sperm concentrations.

In laboratory experiments, the researchers found that one of the low sperm count-associated mutations significantly impaired transcription factor RHOXF2/2B’s ability to regulate its target genes. Molecular modeling suggested that this mutation altered its 3-D structure.

“Spermatogonial stem cells allow men — even in their 70s — to generate sperm and father children,” said Song, who also co-authored the Human Molecular Genetics study. “Our finding that Rhox10 is critical for spermatogonial stem cells, coupled with the finding that human RHOX genes are mutated in infertile men, suggests that mutations in these genes cause human male infertility.”

This conclusion is further underscored by the previous finding that men with abnormal sperm characteristics tend to have RHOX genes excessively marked with chemical tags known as methyl groups. Wilkinson, Song and team are now working to better understand precisely how variations in RHOX transcription factors lead to human infertility.

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Genetic ‘Switch’ Identified As Potential Target For Alzheimer’S Disease

A team at the MRC Clinical Sciences Centre (CSC), based at Imperial College London, has found an important part of the machinery that switches on a gene known to protect against Alzheimer’s Disease.

Working in collaboration with scientists at the Hong Kong University (HKU) and the Erasmus University in Rotterdam, CSC associate professor Richard Festenstein explored the steps by which this Neuroglobin gene is gradually switched on, or up-regulated.

Neuroglobin has previously been shown to protect against Alzheimer’s disease in mice in which it makes the protective Neuroglobin. It is thought that the gene might play a protective role early in the disease in patients, but appears to be down-regulated as the disease progresses. It may therefore prove useful in developing new ways to try to prevent or treat this common cause of dementia, for which there is currently no cure.

Professor Festenstein and Dr Tan-Un from HKU, with help from Professor Sjaak Phillipsen at the Erasmus University, examined how the Neuroglobin gene ‘folds up’ in the cell using a technique called chromosome conformation capture. In results published today in the journal Nucleic Acids Research, they showed that a particular region of DNA, outside the coding region of the Neuroglobin gene itself, loops round to make contact with the start of the gene.

They tested the ability of this newly-identified DNA region to switch on the Neuroglobin gene using two approaches. First, they linked the DNA region directly to another so-called ‘reporter’ gene, and demonstrated simply that it does indeed act as an up-regulator. Second, they used the new ‘Crispr’ technique of gene editing to completely remove this section of DNA from the cell, and showed that the Neuroglobin gene was no longer switched on.

Together, the results gave the team confidence that this newly-identified DNA region is indeed a powerful switching mechanism of the Neuroglobin gene.

As Neuroglobin is thought to be protective in Alzheimer’s, it may be possible in the future to use this ‘switch’ in developing new treatments, such as gene therapy. Such therapeutic approaches require a compact ‘chunk’ of DNA to be most efficient. Importantly, the team pinpointed the position of the new regulatory region, and found that it is some distance away from the Neuroglobin gene itself. It may now be possible to remove the less relevant sections of DNA in between the Neuroglobin gene and its regulator to create an efficient therapeutic gene therapy unit. It may be that this target may prove useful not only in Alzheimer’s but also in other neurodegenerative diseases.

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Uthealth Researchers Identify Genetic Marker For Heart Failure

A team of scientists at The University of Texas Health Science Center at Houston (UTHealth) and Baylor College of Medicine, led by Eric Boerwinkle, Ph.D., Richard Gibbs, Ph.D., and Bing Yu, Ph.D., have identified powerful predictors of congestive heart failure, a major cause of hospitalization and death in the United States. The discovery, published today in Science Advances, was made through an analysis of how gene mutations affect circulating metabolites in the human body.

The human metabolome is a collection of small molecules called metabolites that result from cellular and biological processes in the body and can act as predictors of future disease. Researchers studied how naturally occurring gene mutations can affect metabolites in the genomes of 1,361 African-American participants in the Atherosclerosis Risk in Communities (ARIC) study. ARIC is a longitudinal, population study designed to investigate the origins and predictors of heart disease, stroke and other chronic diseases.

A mutated gene, SLCO1B1, was found to be associated with high levels of blood fatty acid, which is a strong predictor for the development of future heart failure and the mutation itself has a direct effect on heart failure risk.

Because of the aging population, the estimated prevalence and cost of care for heart failure is expected to increase dramatically. By 2030, it’s estimated that more than 8 million people in the United States will have heart failure with $70 billion total costs, according to the American Heart Association. A major risk factor of heart failure is high blood pressure, or hypertension, which is more common among African-Americans.

“The key to heart failure is to identify those at increased risk early. Our hope with this discovery is that we can be more aggressive in treating hypertension if we know someone is genetically predisposed to heart failure,” said Boerwinkle, Kozmetsky Family Chair in Human Genetics and dean of UTHealth School of Public Health.

While the finding was made in a population of African-American participants, the researchers were able to confirm the relationship among European Americans as well.

“African-Americans have higher rates of hypertension, heart failure and mortality. We would expect our findings can help in the prediction and prevention of heart failure among African Americans,” said Yu, assistant professor in the Department of Epidemiology, Human Genetics and Environmental Sciences at UTHealth School of Public Health.

The research builds upon the group’s work on “knockout humans,” which are naturally occurring mutations that inactivate a certain gene. A typical human exome has dozens of these loss-of-function gene variants. Last year, using this technique, the team identified eight new relationships between genes and diseases.

This paper is the first to examine how mutated genes directly affect the metabolome on a genome-wide scale and then go on to influence disease risk. By studying these relationships, the researchers have discovered a new pathway to identify how genes influence disease, according to Boerwinkle.

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Stem Cell Breakthrough Unlocks Mysteries Associated With Inherited And Sometimes Lethal Heart Conditions

Using advanced stem cell technology, scientists from the Icahn School of Medicine at Mount Sinai have created a model of a heart condition called hypertrophic cardiomyopathy (HCM) — an excessive thickening of the heart that is associated with a number of rare and common illnesses, some of which have a strong genetic component. The stem cell lines scientists created in the lab, which are believed to closely resemble human heart tissue, have already yielded insights into unexpected disease mechanisms, including the involvement of cells that have never before been linked to pathogenesis in a human stem-cell model of HCM. The research was published in the journal Stem Cell Reports.

The genetic disorder discussed in the new study is called cardiofaciocutaneous syndrome (CFC), which is caused by a mutation in a gene called BRAF. The condition is rare and affects fewer than 300 people worldwide, according to the National Institutes of Health. It causes abnormalities of the head, face, skin, and major muscles, including the heart.

To learn more about HCM associated with various genetic diseases, Mount Sinai scientists took skin cells from three CFC patients and turned them into highly versatile stem cells, which were then converted into cells responsible for the beating of the heart. This model has relevance for research on several related and more common genetic disorders, including Noonan syndrome, which is characterized by unusual facial features, short stature, heart defects, and skeletal malformations.

“At present, there is no curative option for HCM in patients with these related genetic conditions,” said Bruce D. Gelb, MD, Director of The Mindich Child Health and Development Institute and Professor in the Departments of Pediatrics, Genetics and Genomic Sciences at the Icahn School of Medicine at Mount Sinai. “If our findings are correct, they suggest we might be able to treat HCM by blocking specific cell signals—which is something we know how to do.”

Dr. Gelb says that about 40 percent of patients with CFC suffer from HCM (two of the three study participants had HCM). This suggests a pathogenic connection, though the link has never been fully explored or explained. The primary goal of the current research was to understand the role of a cell-signaling pathway called RAS/MAPK in the cascade of events leading to HCM in patients with CFCs — and by association, with Noonan syndrome, Costello syndrome, and other similar illnesses.

Observing the disease progression in these heart cells, called cardiomyocytes, Dr. Gelb and his team found that some of the changes were caused by interactions with cells that resemble fibroblasts — the same kinds of cells that produce collagen and other proteins. Fibroblasts make up a significant portion of total heart tissue, although it is the cardiomyocytes that are primarily responsible for pumping blood. “These fibroblast-like cells seem to be producing an excess of a protein growth factor called TGF-beta, which, in turn, caused the cardiomyocytes to hypertrophy, or grow larger,” Dr. Gelb said. “We believe this is the first time the phenomenon has been observed using a human induced pluripotent stem cell model of the disease.”

Prior to this observation, Dr. Gelb and his team assumed hypertrophy was “cell autonomous,” meaning intrinsic to the cardiomyocytes themselves. “Based on our cell culture model, we saw that fibroblasts are playing a key role in giving the heart cells the signal that causes them to get big,” Dr. Gelb said. “That was quite unexpected.”

The therapeutic implications may also be profound. “We were able to block TGF-beta in vitro using antibodies that bind to the protein. When we did that, the cardiomyocytes no longer hypertrophy,” Dr. Gelb said. It’s not certain the same effect would be seen in the many clinical cases of HCM that are not influenced by BRAF or the RAS pathway—essentially a chain of cellular proteins that help transmit signals from surface receptors on the cell to DNA in the nucleus –but researchers believe this could be the case.

The bigger surprise, said Dr. Gelb, “is that we may be talking about a signaling circle” in which fibroblasts trigger the release of a growth factor, which causes cardiomyocytes to hypertrophy, which in turn, prompts fibroblasts to release more of the growth factor.” Dr. Gelb didn’t witness this last part of the circle in his stem cell culture, but evidence of fibroblast stimulation has been reported in mouse models that don’t express the RAS mutation. If the circle theory is validated, Dr. Gelb said, there could be new and broad therapeutic interventions for HCM in both RAS and non-RAS contexts. “In theory, at least, a therapy could be useful for both,” he said.

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September Is Thyroid Cancer Awareness Month

September is Thyroid Cancer Awareness Month, and physicians at the Mount Sinai Health System are sharing tips on prevention and urging high-risk groups to get screened.

“High-risk populations are individuals who have a family history of thyroid cancer, and people who have had exposure to radiation, “ said Mike Yao, MD, Associate Professor of Otolaryngology, Icahn School of Medicine at Mount Sinai. “Early detection is critical because thyroid cancer has a good prognosis and high cure rate, and less treatment is required if the cancer is detected at an early stage.”

According to the American Cancer Society, thyroid cancer is on the rise with an estimated 62,450 new cases diagnosed in 2016. In fact, cases among men and women have tripled over the last three decades. Nearly three out of four cases are found in women, and thyroid cancer is commonly diagnosed at a younger age than most adult cancers. The vast majority of thyroid cancer patients don’t experience symptoms, but most types of thyroid cancers can be successfully treated if detected early.

FREE Thyroid Screening: No registration, appointment or preparation required.
The Department of Otolaryngology-Head and Neck Surgery is hosting a free thyroid screening. It takes five minutes per patient, and includes an examination of the neck and an ultrasound screening of the thyroid.
• The Mount Sinai Hospital (Guggenheim Pavilion-Atrium of the Annenberg Building/1468 Madison Avenue at 100th Street) Thursday, September 29, 11:00 am – 2:00 pm

Thyroid Cancer Surveillance
Patients with a diagnosis of papillary thyroid cancer (the most common type) are not always required to pursue surgery. If their tumor is less than one centimeter in diameter, and has favorable characteristics, they have the option to be under strict monitoring with repeat ultrasound imaging to watch for possible growth. If the tumor does not grow, surgery may not be necessary. This management strategy called “active surveillance,” and used in low-risk patients with small cancers, has been endorsed by the American Thyroid Association.

“Every thyroid cancer patient should be treated specific to their disease, and by using this approach, surgery may be avoided in some cases,” said Ilya Likhterov, MD, Assistant Professor of Otolaryngology, Icahn School of Medicine at Mount Sinai. “In many instances, using active surveillance may replace surgery.”

Genetic Testing
Mutational testing for thyroid cancer has been greatly refined over the last few years, and is becoming a more widely available and useful option for patients. If a needle biopsy on a nodule shows that it is not clearly benign or malignant, genetic testing can be done to narrow down the range of risk. It looks for specific mutations in the nodule and gives a more accurate prediction of possible malignancy. This helps patients decide what treatment, if any, should be pursued.

“Mutational testing has been extremely important tool for physicians to counsel patients by giving them hard, numerical data,” said Marita Teng, MD, Residency Program Director, Associate Professor, Department of Otolaryngology, Icahn School of Medicine at Mount Sinai. “We can make better decisions on observing patients with no treatment, or consider surgery sooner rather than later.”

Facts on Thyroid Cancer

• Thyroid cancer is a cancerous tumor or growth located within the thyroid gland
• Thyroid cancer is more common in women, and in people 30 and older
• There are several types of thyroid cancer; the most common is papillary carcinoma which is often curable, especially if caught early
• History of radiation to the head, neck, or chest; exposure to radiation; and family history of thyroid cancer are important risk factors
• Symptoms of thyroid cancer include a lump in the neck by the thyroid, neck pain (sometimes going up the ears), hoarseness in the voice, difficulty swallowing and breathing, persistent cough, and enlarged lymph glands in the neck
• Regular follow-up care is an important part of treatment for patients with thyroid cancers

Tips for Thyroid Cancer Prevention

• Have a physical exam every year
• Have a thyroid physical exam every three years if you are 20-39 years old
• Have a thyroid physical exam every year if you are 40 or older
• Avoid unnecessary exposure to radiation
• Get frequent checks if you’ve been exposed to radiation of the head neck or chest
• Perform a thyroid neck self-exam, looking for asymmetries or protrusions below the Adam’s apple

Experts Available for Interviews

• Marita Teng, MD, Residency Program Director, Associate Professor, Department of Otolaryngology, Icahn School of Medicine at Mount Sinai; member of the Head and Neck Institute at The Mount Sinai Hospital
http://www.mountsinai.org/profiles/marita-s-teng
• Mike Yao, MD, Associate Professor of Otolaryngology, Icahn School of Medicine at Mount Sinai
http://www.mountsinai.org/profiles/mike-yao
• Edward Shin, MD, Chair, Department of Otolaryngology-Head and Neck Surgery, New York Eye and Ear Infirmary of Mount Sinai; Professor of Clinical Otolaryngology, Icahn School of Medicine at Mount Sinai
http://www.mountsinai.org/profiles/edward-j-shin
• Stimson Schantz, MD, Surgeon Director, Medical Board for Otolaryngology, New York Eye and Ear Infirmary of Mount Sinai; Professor of Otolaryngology, Icahn School of Medicine at Mount Sinai
http://www.mountsinai.org/profiles/stimson-p-schantz
• Mark Urken, MD, Co-Director, Institute for Head Neck and Thyroid Cancer, Mount Sinai Beth Israel; Professor of Otolaryngology-Head and Neck Surgery, Icahn School of Medicine at Mount Sinai
http://www.mountsinai.org/profiles/mark-l-urken
• Ilya Likhterov, MD, Assistant Professor of Otolaryngology, Icahn School of Medicine at Mount Sinai
http://www.mountsinai.org/profiles/ilya-likhterov

Patient Available for Interviews

Professional singer and songwriter, 38-year-old Bess McCrary is celebrating her emotional five year milestone of being cancer free, and is excited to share her incredible journey of surviving and thriving after thyroid cancer. McCrary’s encounter with cancer began during a routine cleaning at the dentist in 2011, when a hygienist noticed her enlarged thyroid. With a family history of thyroid cancer, McCrary immediately visited her ENT, and the biopsy of her thyroid nodules came back positive with stage 3 cancer. Terrified by any surgical treatment for her cancer, McCrary understood the possibility of damage to her vocal nerves and the end of her career. She was referred to Dr. Marita Teng at The Mount Sinai Hospital, who performed a successful thyroidectomy to remove her cancer which was followed by radiation, and months of intense vocal therapy at Mount Sinai. Now cancer free, McCrary was determined to sing professionally again and recently recorded and released an album with her “new” voice. She believes Dr. Teng’s care and dedication played a critical role in her success. “It’s that kind of personal care and interest that will keep me and my (growing) family coming back to Mount Sinai for years to come,” said McCrary. She’s still in touch with Dr. Teng and recently told her she gave birth to a baby girl this summer!

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High Expression of Short Gene Appears to Contribute to Destructive Eye Pressures in Glaucoma

Scientists have found a variation of the miR-182 gene in patients with primary open-angle glaucoma that results in this overexpression, said Dr. Yutao Liu, vision scientist and human geneticist in the Department of Cellular Biology and Anatomy at the Medical College of Georgia at Augusta University.

Its impact appears to reduce the ability of the cells in the eye’s trabecular meshwork to continually move the clear aqueous humor out of the front of the eye and dump it into the body’s general circulation, said Liu, corresponding author of the study in the journal Investigative Ophthalmology & Visual Science.

“Every 90 minutes, it turns over,” Liu said of the typically balanced process. Since there is no direct blood supply to the front of the eye, the ciliary body just under the colored portion of the eye uses nearby blood to make the fluid to nourish and oxygenate the area, while trabecular meshwork cells near the cornea scoop it up then dump it – along with waste products – back into the circulation.

In glaucoma, there is a problem with outflow, fluid production or both, which results in increased pressure inside the eyeball that damages the optic nerve and can destroy vision. While current therapies help normalize pressure, they typically only slow disease progression.

In the search to learn more about what causes and could better control the disease, the scientists did a genetic analysis of eye tissues and fluids from the National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database, or NEIGHBORHOOD, consortium of 3,853 people with primary open-angle glaucoma and 33,480 healthy controls.

They consistently found higher expression of miR-182 in the different eye tissues they examined from patients with high-tension glaucoma. For example, miR-182 expression in the aqueous humor was twofold higher in high-tension glaucoma patients than controls. Liu notes that since all glaucoma patients were under treatment, their medication’s impact on those levels could not be ruled out, but he suspects that increased expression is a hallmark of the disease state.

Liu recently received a two-year grant from the Bright Focus Foundation (brightfocus.org/ ) that is enabling next-step studies such as exploration of the impact of current drug therapy on miR-182. Elevated miR-182 expression already is associated with premature aging of the eye and, when MCG scientists mimic premature aging by applying hydrogen peroxide, miR-182 levels increase.

Now, they are also are looking at the impact on the trabecular meshwork cells’ ability to take up aqueous humor and deliver it back to the circulation.

Adding color to the fluid is easing their ability to watch its transport, and 3-D imaging is providing additional insight into how the cells are functioning. The additional insight will help answer questions like whether trabecular meshwork cells become stiff and less capable of making the pores that let fluid come onboard for their endless transport task as Liu suspects. He’s also looking at the impact on the ability of trabecular meshwork cells to make and send out fluid-filled pockets called exosomes that may enable the cells to communicate.

Further genetic analysis will also enable identification of additional targets of miR-182. “If we find some targets that constantly change in the eye with glaucoma, then we may be able to find some small chemicals to target those genes in the eye,” he said of these “druggable” targets. Longer-term goals are therapies that more directly target the major pathway in this typically age-related disease.

One known miR-182 target is CHEK2, a known tumor suppressor and suspected genetic risk factor for high-tension glaucoma. CHEK2 has been associated with an enlargement of the tip of the optic nerve that indicates high-pressure damage.

Mutations of miR-182 have been associated with a wide range of disorders, including insomnia and depression, and elevated expression appears to impact genes that regulate the body’s circadian rhythm. It also appears to have a wide impact on normal body regulation from DNA repair to the immune system. In the eye, it’s normally highly expressed in the retina, where it appears to play a role in normal visual function, but has not been clearly associated with disease in this back portion of the eye.

A microRNA, or miRNA, like miR-182 is a small non-coding RNA molecule, which means, rather than actually producing proteins like regular DNA, it helps regulate protein production. There are several hundred of these in each tissue of the body, Liu said.

Known risk factors for glaucoma in addition to age and elevated intraocular pressure, include family history, African ancestry and genetic factors. Primary open-angle glaucoma affects about 1 percent of Americans, according to The Glaucoma Foundation. A percentage of those patients have normal-tension glaucoma, experiencing classic problems such as optic nerve damage and vision loss, despite normal eye pressures. The majority of patients with the miR-182 variation in the study had high-tension glaucoma, Liu noted.

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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.

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Similarities Unite Three Distinct Gene Mutations of Treacher Collins Syndrome

Scientists at the Stowers Institute for Medical Research have reported a detailed description of how function-impairing mutations in polr1c and polr1d genes cause Treacher Collins syndrome (TCS), a rare congenital craniofacial development disorder that affects an estimated 1 in 50,000 live births.

Collectively the results of the study, published in the current issue of PLoS Genetics, reveal that a unifying cellular and biochemical mechanism underlies the etiology and pathogenesis of TCS and its possible prevention, irrespective of the causative gene mutation.

Loss-of-function mutations in three human genes, <i>TCOF1, POLR1C and POLR1D, have been implicated in TCS and are thought to be responsible for about 90 percent of the diagnoses of this congenital craniofacial condition.

The clinical manifestations of TCS include facial anomalies such as small jaws and cleft palate, hearing loss, and respiratory problems. Patients with TCS typically undergo multiple surgeries, but rarely are they fully corrective. By uncovering a mechanism of action common to all three genes, Stowers scientists have advanced scientific understanding of TCS etiology and pathogenesis and identified possible new avenues for preventing or treating the birth defect. This latest study from the laboratory of Stowers Investigator Paul Trainor, Ph.D., focused on Polr1c and Polr1d, whose roles as a genetic cause of TCS were revealed in a 2011 study of a small group of patients who had been diagnosed with TCS but who did not have the TCOF1 mutation. Unlike POLR1C and POLR1D, TCOF1 has been long recognized as a causative gene in TCS and as a result has been more extensively investigated.

“Before we began the study, nothing was known about the role of Polr1c and Polr1d in craniofacial development,” said Kristin Watt, Ph.D., lead author of the PLoS Genetics paper and postdoctoral scientist in the Trainor lab. “Using zebrafish as our animal model, we set out to explore the functional roles of polr1c and polr1d during embryogenesis and more specifically in craniofacial development.”

Trainor, Watt and their collaborators compared the results of their findings on polr1c and polr1d with their and other labs’ previous research results on Tcof1. In all three loss-of-function models, the researchers found that the chain of cellular events that led to the TCS phenotype of abnormal craniofacial development originated in ribosomes, the cellular components that translate messenger RNA into proteins. Like the Tcof1 gene, polr1c and polr1d mutations were found to perturb ribosome biogenesis, or production of ribosomes, which affects the generation and survival of progenitor neural crest cells, the precursors of craniofacial bone, cartilage and connective tissue.

In animal models of all three causative genes, the scientists determined that deficient ribosome biogenesis triggered a p53-dependent cell death mechanism in progenitor neural crest cells. As a result of the activation of the p53 gene, developing embryos no longer made the quantity of neural crest cells needed to properly form the craniofacial skeleton.

However, in the polr1c and polr1d models as in the Tcof1 models, Stowers scientists found that by experimentally blocking p53 activation, they could restore the neural crest cell population and thereby rescue the animal models’ cranioskeletal cartilage.

Despite the rescue effect, Trainor said that he does not view the “guardian of the genome,” as the p53 gene is often called due to its ability to suppress cancer, as the basis of a potential therapy to prevent or reduce TCS during embryonic development. The p53 gene’s association with cancer makes inhibiting its function too risky, he said.

A less risky and perhaps more effective target for the prevention or treatment of TCS could be enhancing ribosomes, Trainor said, because the loss-of-function mutations in all three causative genes involve ribosome RNA (rRNA) transcription. Polr1c and Polr1d, for example, are subunits of RNA polymerases I and III that are essential for ribosome biogenesis.

“Rather than blocking p53, a better approach may be to try to prevent TCS by treating the problem in ribosome biogenesis that triggers the activation of p53 and the loss of neural crest cells,” said Trainor.

In their research with zebrafish embryos, Trainor and collaborators also determined that polr1c and polr1d are spatiotemporally and dynamically expressed, particularly during craniofacial development. Furthermore, zebrafish embryos with the polr1c and polr1d loss-of-function mutations develop abnormalities in craniofacial cartilage development that mimic the clinical manifestations of TCS in patients. Trainor said that he and his fellow researchers were surprised that mutations in polr1c and polr1das well as Tcof1 specifically affected craniofacial development, because ribosome biogenesis occurs in every cell of the body. The mutation of a gene that is part of the ribosome complex would be expected to be detrimental to each of these cells, he said. However, in the zebrafish models, the mutation appears to primarily affect progenitor neural crest cells. Trainor said that he and his team theorize that progenitor neural crest cells may be particularly sensitive to deficiencies in ribosome biogenesis during embryogenesis.

Thus, the study revealed new animal models for TCS: zebrafish with polr1c and polr1d loss-of-function mutations. Moreover, the existence of a common mechanism of action may simplify the research, particularly the search for a therapy to prevent or treat TCS. Because of the similarities among the three causative genes, “we may be able to develop creative ways of preventing TCS that will prove effective in at risk individuals who have one of the gene mutations,” said Trainor, who has investigated the molecular origins and development of TCS and related craniofacial developmental disorders for 10 years.

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Researchers ID Cancer Gene-Drug Combinations Ripe for Precision Medicine

In an effort to expand the number of cancer gene mutations that can be specifically targeted with personalized therapies, researchers at University of California San Diego School of Medicine and Moores Cancer Center looked for combinations of mutated genes and drugs that together kill cancer cells. Such combinations are expected to kill cancer cells, which have mutations, but not healthy cells, which do not. The study, published July 21 in Molecular Cell, uncovered 172 new combinations that could form the basis for future cancer therapies.

“Oncologists here at Moores Cancer Center at UC San Diego Health and elsewhere can often personalize cancer therapy based on an individual patient’s unique cancer mutations,” said senior author Trey Ideker, PhD, professor of genetics at UC San Diego School of Medicine. “But the vast majority of mutations are not actionable — that is, knowing a patient has a particular mutation doesn’t mean there’s an available therapy that targets it. The goal of this study was to expand the number of mutations we can pair with a precision therapy.”

Most cancers have gene mutations that do one of two things — promote cell growth or prevent cell death. The first type is the target of many therapies, which inhibit cell growth. But it’s much harder to develop therapies that restore malfunctioning genes that should be triggering cell death in abnormal cells, known as tumor-suppressor genes.

Rather than target a tumor-suppressor gene directly, Ideker and team took the approach of identifying genetic interactions between a tumor suppressor gene and another gene, such that simultaneous disruption of both genes selectively kills cancer cells.

The researchers first used yeast to quickly and cheaply screen 169,000 interactions between yeast versions of human tumor-suppressor genes and genes that can be inhibited with drugs, sometimes called “druggable” targets. To do this, they deleted each gene one at a time, in combination with another mutation. Those experiments whittled down the best combinations — those lethal to the yeast cells — to a few thousand.

Next the team prioritized 21 drugs for which the yeast druggable targets were involved in the greatest number of cell-lethal interactions. They tested these drugs one at a time for lethal interaction with 112 different tumor-suppressor gene mutations in human cancer cells growing in the lab.

The researchers ended up with 172 drug-gene mutation combinations that successfully killed both yeast and human cancer cells. Of these combinations, 158 had not been previously discovered.

Here’s one example of how this information might be useful for doctors and patients: Irinotecan is a drug only indicated by the FDA for use in colon cancer. But this study suggests that this class of drugs should be evaluated for efficacy in any tumor with a mutation that inhibits RAD17, a tumor-suppressor gene that normally helps cells fix damaged DNA.

The next steps will be to test these combinations in more human cancer cell types and eventually in mouse models. But 172 combinations is a lot, more than a single lab can test, the researchers say. They hope other research teams will also take their list and further test each combination in a variety of conditions. To help spread this information to scientists around the world, all of the data from this study has been made freely available on NDEx, a new network data-sharing resource developed by Ideker and UC San Diego School of Medicine data scientist Dexter Pratt.

“We’ve created an important translational research resource for other scientists and oncologists,” said co-first author John Paul Shen, MD, clinical instructor and postdoctoral fellow at UC San Diego School of Medicine and Moores Cancer Center. “And since many of the cancer-killing interactions we discovered involve already FDA-approved drugs, it may mean they could reach clinical translation rapidly. If these results are validated in subsequent testing, in the future an oncologist will have many more options for precision cancer therapy.”