TAG: "Drugs"

Sushi meets science: Mapping the ‘wasabi receptor’

Protein’s structure will guide hunt for new treatments of inflammation-induced pain.

By Pete Farley, UC San Francisco

In a feat that would have been unachievable only a few years ago, researchers at UC San Francisco have pulled aside the curtain on a protein informally known as the “wasabi receptor,” revealing at near-atomic resolution structures that could be targeted with anti-inflammatory pain drugs.

Officially named TRPA1 (pronounced “trip A1”), the newly visualized protein resides in the cellular membrane of sensory nerve cells. It detects certain chemical agents originating outside our bodies — pungent irritants found in substances ranging from wasabi to tear gas — but is also triggered by pain-inducing signals originating within, especially those that arise in response to tissue damage and inflammation.

“The pain system is there to warn us when we need to avoid things that can cause injury, but also to enhance protective mechanisms,” said David Julius, Ph.D., professor and chair of UCSF’s Department of Physiology, and co-senior author of the new study, which appears in the April 8, 2015 online issue of Nature. “We’ve known that TRPA1 is very important in sensing environmental irritants, inflammatory pain, and itch, and so knowing more about how TRPA1 works is important for understanding basic pain mechanisms. Of course, this information may also help guide the design of new analgesic drugs.”

TRPA1 receptor proteins form pores called ion channels in sensory nerve cell membranes. These channels, normally closed, open in response to certain chemical signals, which allows ions to pass into the cell’s interior, triggering a warning impulse. But without knowing enough about the receptor’s overall structure to see where a given compound might connect, designing a drug to alleviate pain by controlling the action of the ion channel is something of a shot in the dark.

Julius and co-senior author Yifan Cheng, Ph.D., associate professor of biochemistry and biophysics, were able to capture images of TRPA1 that reveal its structure in three dimensions, including a cleft in which an experimental drug molecule sits when it binds to the channel. “A few drugs have been developed that target TRPA1, and in our 3-D structure we can see where one such drug binds,” said Julius. “This provides important insight into how this one major class of drugs interacts with TRPA1 and thus how it may work to block channel function.”

UCSF postdoctoral fellows and co-first authors Candice Paulsen, Ph.D., and Jean-Paul Armache, Ph.D., spearheaded the TRPA1 project. Yuan Gao, a graduate student in Cheng’s lab, also took part in the work. The team used an approach called electron cryo-microscopy (cryo-EM), an imaging technique in which proteins are bombarded with electrons at very low temperatures.

Thanks to a number of innovative hardware and software advances — some developed at UCSF by Cheng and David Agard, Ph.D., professor of biophysics and biochemistry and a Howard Hughes Medical Institute investigator — cryo-EM has undergone a revolution in image quality over the past several years. Using these tools, the group imaged TRPA1 at a resolution of about 4 angstroms. By way of comparison, the thickness of a credit card is about 8 million angstroms.

Julius and Cheng began their cryo-EM collaborations about six years ago when they were in pursuit of the structure of a related channel called TRPV1. Sometimes called the capsaicin receptor, TRPV1 can be triggered by the chemical that lends “heat” to chili peppers, but it also responds to actual heat when temperatures reach uncomfortably high levels. TRPV1 was the first protein of such small size to be imaged to near-atomic resolution by cryo-EM, work that was reported in Nature in December 2013.

The determination of TRPV1’s structure by cryo-EM “sent shockwaves through the field of structural biology,” Cheng said, because many researchers had dismissed the method as “blob-ology”: until quite recently cryo-EM’s resolution — about 15 angstroms at best — was far too coarse to discern the subtleties of structure in molecules as small as TRP ion channels.

For decades, X-ray crystallography, which involves first coaxing a protein of interest to crystallize, and then analyzing diffraction patterns created as X-rays pass through the crystal, has been the standard method of determining molecular structures. While crystallography can attain resolutions below 3 angstroms, it requires large quantities of a protein. Moreover, obtaining a usable crystal can take years, and many biologically important proteins — especially membrane proteins, which are crucial to cellular signaling — have never been successfully crystallized.

TRP channels were among these, so “we came at it from a different angle,” said Cheng. “Since crystallization was so difficult, we decided to try cyo-EM, which bypasses this requirement.”

For cryo-EM, the proteins of interest are placed in an aqueous solution, then frozen in a very thin sheet of ice, so quickly that the water doesn’t have time to form crystals. It is critical that the ice remains in a glassy state, because formation of any ice crystals would damage proteins embedded within ice and interfere with determining the structure of the proteins in their native conformation.

With many copies of the proteins suspended in this glassy ice, like insects trapped in amber, the researchers capture as many as 100,000 images, then computationally combine these thousands of two-dimensional views to generate the three-dimensional structure of the protein.

The images Julius and Cheng’s group produced for the TRPA1 ion channel show it in three different states — closed, open, and partially open — a range that offers a lot of insight into the channel’s workings. Though the images of TRPA1 generated in the new study represent only slightly different conformations, the scientists expect to generate additional structures in more distinct conformations in future research.

The research was supported by the National Institutes of Health and the UCSF Program for Breakthrough Biomedical Research. Co-first author Paulsen was supported by a Postdoctoral Training Grant from the UCSF Cardiovascular Research Institute, and is currently a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation.

“Cryo-EM has undergone a ‘resolution revolution’ that has enabled us to literally see TRP channels in all their glory,” said Julius. “We’ve had some idea what TRPA1 might look like, but there’s something elegant and satisfying about obtaining the structure, because seeing really is believing.”

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NPR: Sushi science: A 3-D view of the body’s wasabi receptor

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Dual therapy’s one-two punch knocks out drug-resistant lung cancer

Unusual chance to study patient’s residual tumor leads to new finding.

PET-CT imaging from a patient with EGFR-mutant lung cancer before (top) and after (bottom) treatment with the targeted drug erlotinib (Tarceva). By studying the residual tumor seen in the lower image, a UCSF team determined why the patient developed erlotinib resistance, and devised a strategy to overcome it. (Image courtesy of Collin Blakely)

By Pete Farley, UC San Francisco

Capitalizing on a rare opportunity to thoroughly analyze a tumor from a lung cancer patient who had developed resistance to targeted drug treatment, UC San Francisco scientists identified a biological escape hatch that explains the resistance, and developed a strategy in mice for shutting it down.

In experiments that combined the drug the patient had taken with a second compound that blocks off this newly discovered resistance pathway, the researchers were able to durably wipe out cancer cells in mice implanted with cells from the drug-resistant tumor.

“Even in cancers that are responding to targeted therapy by conventional criteria, resistance is already developing,” said the senior author of the new study, Trever Bivona, M.D., Ph.D., assistant professor of medicine and member of the UCSF Helen Diller Family Comprehensive Cancer Center (HDFCCC). “In this work we have begun to crack open the question of why residual disease persists after targeted therapy.”

Between 10 and 35 percent of non-small cell lung cancer (NSCLC) patients carry mutations in a gene that codes for a cell-surface protein called the epidermal growth factor receptor, or EGFR. As its name suggests, under normal circumstances, when a growth factor protein locks onto the EGFR, the receptor sends signals that prompt cells to divide and proliferate. But the EGFR mutations seen in NSCLC cause the receptor to be stuck in an “on” position, leading to rampant cell proliferation.

Over the past decade, medications such as erlotinib (trade name Tarceva), which precisely targets the EGFR and tamps down its activity, have advanced the treatment of EFGR-mutant NSCLC beyond chemotherapy, but significant challenges remain. As many as 30 percent of patients exhibit so-called primary resistance to EGFR inhibitors, in which the drugs have no detectable effect. And among patients who do respond, almost all have an incomplete response leading to acquired resistance, in which drug-resistant cells that survive treatment form residual, often lethal, tumors.

Understanding the biological basis of acquired resistance has proved difficult, partly because patients with late-stage lung cancer rarely undergo surgery, leaving scientists with few drug-resistant tumors to use in research. But as described in the online edition of Cell Reports today (April 2), a team of UCSF researchers recently had unusual access to a surgically resected tumor from an EGFR-mutant lung cancer patient who had experienced a substantial, but incomplete, response to erlotinib.

Led by first authors Collin Blakely, M.D., Ph.D., a clinical instructor at UCSF, and Evangelos Pazarentzos, Ph.D., a postdoctoral fellow, the research group analyzed cells from this tumor using next-generation genome sequencing in an effort to understand how the cells sidestepped erlotinib treatment. They found that the tumor cells retained the EGFR mutation targeted by erlotinib and had not acquired additional cancer-driving mutations, or any other mutations known to confer drug resistance. These results suggested that the cells were still potentially susceptible to erlotinib, but had enlisted some additional mechanism to survive treatment.

That mechanism was revealed when cells from the tumor were implanted in mice that were then treated with erlotinib. The drug effectively inhibited EGFR activity, but the researchers also observed a rapid, 10-fold increase in the activity of a pathway known as NF-kappa-B, and they discovered that this increase is mediated by a previously unknown biochemical complex formed within the tumor cells. Though primarily associated with the immune system, a growing body of work has tied the NF-kappa-B pathway to various forms of cancer.

An experimental drug known as PBS-1086 directly targets the NF-kappa-B pathway, and when the researchers coupled this drug with erlotinib, the implanted tumors shrank significantly, suggesting that combining a compound like PBS-1086 with erlotinib at the outset of therapy may help to prevent acquired drug resistance in EGFR-mutant NSCLC.

Combined drug regimens designed to overcome drug resistance at the outset of therapy are now the norm in treating certain forms of melanoma, said Bivona, and he believes PBS-1086 “has a shot” to play a similar role in NSCLC.

“The NF-kappa-B pathway is engaged by cells in response to EGFR inhibitors as a way to survive treatment,” Bivona said. “Excitingly, if we block that pathway with a novel drug while simultaneously administering the EGFR inhibitor, we see tumors shrink. In lung cancer patients treated with these drugs, and that’s a substantial number of patients, this could be a very powerful companion therapy to minimize or eliminate residual disease.”

Other HDFCCC researchers taking part in the research included Sourav Bandyopadhyay, Ph.D., assistant professor of bioengineering and therapeutic sciences, and Nevan J. Krogan, Ph.D., professor of cellular and molecular pharmacology.

The work was funded by the Bonnie J. Addario Lung Cancer Foundation; the National Institutes of Health; the Howard Hughes Medical Institute; the Doris Duke Charitable Foundation; the American Lung Association; the Sidney Kimmel Foundation for Cancer Research; the Searle Scholars Program; the California Institute for Quantitative Biosciences; and the Li Ka-shing Foundation.

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Nanotech platform shows promise for treating pancreatic cancer

Researchers create new method that may solve some problems of using chemotherapy.

Andre Nel, UCLA

By Shaun Mason, UCLA

Scientists at UCLA’s California NanoSystems Institute and Jonsson Comprehensive Cancer Center have combined their nanotechnology expertise to create a new treatment that may solve some of the problems of using chemotherapy to treat pancreatic cancer.

The study, published online in the journal ACS Nano, describes successful experiments to combine two drugs within a specially designed mesoporous silica nanoparticle that looks like a glass bubble. The drugs work together to shrink human pancreas tumors in mice as successfully as the current standard treatment, but at one-twelfth the dosage. This lower dosage could reduce both the cost of treatment and the side effects that people suffer from the current method.

The study was led by Dr. Huan Meng, assistant adjunct professor of medicine, and Dr. Andre Nel, distinguished professor of medicine, both at the Jonsson Cancer Center.

Pancreatic cancer, a devastating disease with a five-year survival rate of 5 percent, is difficult to detect early and symptoms do not usually appear until the disease is advanced. As a result, many people are not diagnosed until their tumors are beyond the effective limits of surgery, leaving chemotherapy as the only viable treatment option. The chemotherapy drug most often used for pancreas cancer is gemcitabine, but its impact is often limited.

Recent research has found that combining gemcitabine with another drug called paclitaxel can improve the overall treatment effect. In the current method, Abraxane — a nano complex containing paclitaxel — and gemcitabine are given separately, which works to a degree, but because the drugs may stay in the body for different lengths of time, the combined beneficial effect is not fully synchronized.

“The beauty of the silica nanoparticle technology is that gemcitabine and paclitaxel are placed together in one special lipid-coated nanoparticle at the exact ratio that makes them synergistic with one another when co-delivered at the cancer site, giving us the best possible outcome by using a single drug carrier,” Meng said. “This enables us to reduce the dose and maintain the combinatorial effect.”

After the scientists constructed the silica nanoparticles, they suspended them in blood serum and injected them into mice that had human pancreas tumors growing under their skin. Other mice with tumors were given injections of saline solution (a placebo with no effect), gemcitabine (the treatment standard), and gemcitabine and Abraxane (an FDA-approved combination shown to improve pancreas cancer survival in humans).

In the mice that received the two drugs inside the nanoparticle, pancreas tumors shrank dramatically compared with those in the other mice.

Similar comparisons were made with mouse models, in which the human tumors were surgically implanted into the mice’s abdomens in order to more closely emulate the natural point of origin of pancreatic tumors and provide a better parallel to the tumors in humans. In these experiments, the tumors in the mice receiving silica nanoparticles shrank more than the comparative controls. Also, metastasis, or tumor spread, to nearby organs was eradicated in these mice.

“Instead of just a laboratory proof-of-principle study of any cancer, we specifically attacked pancreatic cancer with a custom-designed nanocarrier,” said Nel, who is also associate director for research of the California NanoSystems Institute. “In our platform, the drugs are truly synergistic because we have control over drug mixing, allowing us to incorporate optimal ratios in our particles, making the relevance of our models very high and our results very strong.”

Meng said the silica nanocarrier must still be refined for use in humans. The researchers hope to test the platform in human clinical trials within the next five years.

The research was supported by the U.S. Public Health Service and the National Science Foundation.

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UCLA researchers develop new combination therapy to fight melanoma

Triple combination therapy helps immune system attack cancerous tumors.

By Reggie Kumar, UCLA

UCLA cancer researchers have found that a new triple combination therapy shows promising signs of more effectively controlling advanced melanoma than previous treatments and also with fewer side effects.

An estimated 70,000 new cases of metastatic melanoma are diagnosed each year in the United States, and of those 8,000 people will die of the disease. About 50 percent of the total men and women diagnosed have a mutated protein called a BRAF mutation, which in most cases allows melanoma to eventually build up a resistance to many drug therapies.

In the new study led by UCLA Jonsson Comprehensive Cancer Center member Dr. Antoni Ribas and colleague Dr. Siwen Hu-Lieskovan, UCLA scientists combined targeted therapies utilizing the BRAF inhibitor drug dabrafenib and the MEK inhibitor drug trametinib, with immunotherapy, which is treatment that uses a person’s own immune system to help fight cancer.

Dabrafenib causes cancerous tumors to shrink in people whose metastatic melanoma has a BRAF gene change. Trametinib prevents the disturbance of the MAPK/ERK pathway that dabrafenib causes on cells without the BRAF mutation. That disturbance causes overactive cells to form a different type of skin cancer.

The combination of the three therapies, which was shown to be a more effective treatment, works by sensitizing a person’s own immune system to enhance immunotherapy, and reducing the probability of the melanoma eventually developing resistance.

This is a significant advance compared to previous drug combination findings, in which a combination of the BRAF inhibitor vemurafenib with an immunotherapy drug caused serious liver toxicity in some people, and the targeted therapies (BRAF and/or MEK inhibitors) became less effective and reactivated cancer cell growth.

“The two-drug combination of BRAF and MEK inhibitors works synergistically and decreases the side effects of the BRAF inhibitor on normal cells. We reasoned that this combo would allow us to synergize with immunotherapy without increasing toxicities,” said Ribas, a professor of hematology-oncology at UCLA. “We have made incredible progress in the last three years of treating advanced melanoma, with six new drug therapies approved by the FDA. Half are immunotherapies and the other half are BRAF or MEK inhibitors. The next step is to figure out how to combine them and merge their benefits in the clinic.”

The triple combination of targeted therapies dabrafinib and trametinib inhibitors with immunotherapy (tumor antigen-specific adoptive cell transfer or anti-PD1 antibody) makes immune therapy more effective at killing cancerous tumors and it causes less toxicity, said Hu-Lieskovan, a UCLA clinical instructor of hematology and oncology.

“We’re trying to take advantage of the high response rate of the targeted therapy and durability of the immune therapy to induce a response that lasts in the majority of patients,” Hu-Lieskovan said.

Ribas and Hu-Lieskovan have opened two clinical trials to test the effectiveness of the triple combination therapy in advanced melanoma patients. The first reported findings will be presented at the American Society of Clinical Oncology annual meeting in May.

The study is available online today (March 18) in the journal Science Translational Medicine.

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Bioengineers put human hearts on chip to aid drug screening

Chips ultimately could replace use of animals to screen drugs for safety, efficacy.

By Sarah Yang, UC Berkeley

When UC Berkeley bioengineers say they are holding their hearts in the palms of their hands, they are not talking about emotional vulnerability.

Instead, the research team led by bioengineering professor Kevin Healy is presenting a network of pulsating cardiac muscle cells housed in an inch-long silicone device that effectively models human heart tissue, and they have demonstrated the viability of this system as a drug-screening tool by testing it with cardiovascular medications.

This organ-on-a-chip, reported in a study published today (March 9) in the journal Scientific Reports, represents a major step forward in the development of accurate, faster methods of testing for drug toxicity. The project is funded through the Tissue Chip for Drug Screening Initiative, an interagency collaboration launched by the National Institutes of Health to develop 3-D human tissue chips that model the structure and function of human organs.

“Ultimately, these chips could replace the use of animals to screen drugs for safety and efficacy,” said Healy.

The study authors noted a high failure rate associated with the use of nonhuman animal models to predict human reactions to new drugs. Much of this is due to fundamental differences in biology between species, the researchers explained. For instance, the ion channels through which heart cells conduct electrical currents can vary in both number and type between humans and other animals.

“Many cardiovascular drugs target those channels, so these differences often result in inefficient and costly experiments that do not provide accurate answers about the toxicity of a drug in humans,” said Healy. “It takes about $5 billion on average to develop a drug, and 60 percent of that figure comes from upfront costs in the research and development phase. Using a well-designed model of a human organ could significantly cut the cost and time of bringing a new drug to market.”

The heart cells were derived from human-induced pluripotent stem cells, the adult stem cells that can be coaxed to become many different types of tissue.

The researchers designed their cardiac microphysiological system, or heart-on-a-chip, so that its 3-D structure would be comparable to the geometry and spacing of connective tissue fiber in a human heart. They added the differentiated human heart cells into the loading area, a process that Healy likened to passengers boarding a subway train at rush hour. The system’s confined geometry helps align the cells in multiple layers and in a single direction.

Microfluidic channels on either side of the cell area serve as models for blood vessels, mimicking the exchange by diffusion of nutrients and drugs with human tissue. In the future, this setup also could allow researchers to monitor the removal of metabolic waste products from the cells.

“This system is not a simple cell culture where tissue is being bathed in a static bath of liquid,” said study lead author Anurag Mathur, a postdoctoral scholar in Healy’s lab and a California Institute for Regenerative Medicine fellow. “We designed this system so that it is dynamic; it replicates how tissue in our bodies actually gets exposed to nutrients and drugs.”

Within 24 hours after the heart cells were loaded into the chamber, they began beating on their own at a normal physiological rate of 55 to 80 beats per minute.

The researchers put the system to the test by monitoring the reaction of the heart cells to four well-known cardiovascular drugs: isoproterenol, E-4031, verapamil and metoprolol. They used changes in the heart tissue’s beat rate to gauge the response to the compounds.

The baseline beat rate for the heart tissue consistently fell within 55 to 80 beats per minute, a range considered normal for adult humans. They found that the responses after exposure to the drugs were predictable. For example, after half an hour of exposure to isoproterenol, a drug used to treat bradycardia (slow heart rate), the beat rate of the heart tissue increased from 55 to 124 beats per minute.

The researchers noted that their heart-on-a-chip could be adapted to model human genetic diseases or to screen for an individual’s reaction to drugs. They also are studying whether the system could be used to model multi-organ interactions. A standard tissue culture plate could potentially feature hundreds of microphysiological cell culture systems.

“Linking heart and liver tissue would allow us to determine whether a drug that initially works fine in the heart might later be metabolized by the liver in a way that would be toxic,” said Healy.

The engineered heart tissue remained viable and functional over multiple weeks. Given that time, it could be used to test various drugs, Healy said.

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Childhood leukemia study reveals disease subtypes, new treatment option

1 of 8 patients might benefit from highly successful lymphoma drugs.

By Pete Farley, UC San Francisco

A new study of acute lymphoblastic leukemia (ALL), a blood cancer that primarily affects young children, has revealed that the disease has two distinct subtypes, and provides preliminary evidence that about 13 percent of ALL cases may be successfully treated with targeted drugs that have proved highly effective in the treatment of lymphomas in adults.

Usually emerging in children between 2 and 5 years of age, ALL occurs when the proliferation of white blood cells known as lymphocytes spirals out of control. The current standard of care for ALL employs high doses of chemotherapy that usually cure the disease, but may also have serious long-term effects on brain development, bone growth and fertility, so there is an unmet need for better therapies.

In addition to discovering the two ALL subtypes, the researchers, led by scientists from UC San Francisco and Oregon Health & Science University (OHSU), developed a simple lab test that determines whether patients fall into the less-common subtype that may respond to targeted therapy. One author of the new study, affiliated with MD Anderson Cancer Center in Texas, is already using this new test to recruit patients for a phase one clinical trial evaluating the use of targeted drugs for ALL.

The research and resulting clinical trial exemplify one of the main goals of precision medicine — improving health by identifying subtypes of disease that can be specifically targeted with drugs or other therapies.

“We hope patients in this newly identified subset can be treated with these targeted drugs, which have worked very well in patients with lymphoma and which are powerfully effective in the mouse experiments we have conducted on ALL,” said co-senior author Markus Müschen, M.D., Ph.D., professor of laboratory medicine at UCSF and a member of the UCSF Helen Diller Family Comprehensive Cancer Center (HDFCCC). “These drugs have essentially no side-effects and relatively few effects on quality of life.”

Müschen said the new work, reported online in Cancer Cell today (March 9), grew out of a line of research on new treatments for lymphoma, which usually affects adults. That work, which culminated in papers published in The New England Journal of Medicine in 2013, showed that various forms of lymphoma respond well to treatment with ibrutinib (trade name Imbruvica) or idelalisib (trade name Zydelig), two drugs that precisely target the B-cell antigen receptor, a protein found in white blood cells.

“Because B-cells are also involved in ALL, we essentially recapitulated these studies, starting out with the basic science by studying genetic components of the B-cell antigen receptor in mice,” said Müschen. “We were surprised to find that, depending on the initial cancer-causing mutation, B-cell antigen receptor signaling is sometimes present in ALL, which suggested that ALL might also respond to the drugs that had been used in lymphoma.”

Led by first authors Huimin Geng, Ph.D., assistant professor of laboratory medicine at UCSF, postdoctoral fellow Christian Hurtz, Ph.D., also of UCSF, and Kyle Lenz, research assistant at OHSU, the group found that cells that exhibit B-cell antigen receptor signaling also express very high levels of a protein known as BCL-6. Then, using BCL-6 as a biomarker, the team used several methods to inhibit B-cell antigen receptor signaling, including treating cells with targeted compounds used in human lymphoma. All of these approaches successfully and selectively killed ALL cells, and similar results were seen in a mouse model of ALL.

The research group next studied 830 patients enrolled in four ongoing ALL clinical trials, in part to assess whether testing for BCL-6 expression would be a practical biomarker in the clinic to identify candidates for targeted therapy.

Virtually all of the bone marrow slices from 112 patients (13.5 percent) with active B-cell antigen receptor signaling showed “beautiful staining” of BCL-6 expression, Müschen said (in two patients only weak staining was seen). On the other hand, no BCL-6 staining was observed in patients lacking B-cell antigen receptor signaling. These results suggest that the BCL-6 test may have sufficient sensitivity and specificity to select patients for targeted therapy.

“Children are given high doses of chemotherapy for ALL because they are considered more resilient than adults, but there are long-term consequences that may not be obvious in childhood,” Müschen said. “Our idea is that by adding these new drugs we can reduce the amount of conventional chemotherapy or even replace it. In our experiments with mice, both combination therapy with low-dose chemotherapy and single-agent targeted therapy each worked very well. The new clinical trial using the BCL-6 biomarker should begin to bring us the answers.”

OHSU’s Bill Chang, M.D., Ph.D., was co-senior author of the study. The work was funded by the National Institutes of Health, the National Cancer Institute, the Hyundai Hope on Wheels program, the St. Baldrick’s Foundation, the Leukemia and Lymphoma Society, Tucker’s Toy Box Foundation, the William Lawrence and Blanche Hughes Foundation, the California Institute for Regenerative Medicine, and the U.K.’s Medical Research Council and National Institute for Health Research.

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Abnormal brain rhythms tield to problems with thinking in schizophrenia

UCSF study of unique mouse model shows cognitive deficits may be reversible.

By Pete Farley, UC San Francisco

By studying specially bred mice with specific developmental and cognitive traits resembling those seen in schizophrenia, UC San Francisco researchers have provided new evidence that abnormal rhythmic activity in particular brain cells contributes to problems with learning, attention and decision-making in individuals with that disorder.

As reported in today’s (March 5) online edition of Neuron, when the researchers corrected these cells’ faulty rhythm, either by directly stimulating the cells or by administering low doses of a commonly used drug, cognitive deficits in the mice were reversed, results that point the way to possible therapies to address cognitive symptoms in individuals with schizophrenia.

According to Vikaas Sohal, M.D., Ph.D., senior author of the new study, in addition to dealing with the burdens of schizophrenia’s so-called positive symptoms (such as delusions and hallucinations) and negative symptoms (such as social withdrawal and a lack of motivation), individuals with the disorder also grapple with cognitive deficits that create considerable challenges in the arenas of education, work and interpersonal relationships.

Converging evidence from many previous studies has implicated a population of neurons in the brain’s prefrontal cortex called fast-spiking (FS) interneurons in schizophrenia, but a causal relationship between malfunctioning FS interneurons and cognitive symptoms of the disorder has not yet been firmly established, Sohal said.

For example, individuals with schizophrenia perform poorly on the Wisconsin Card-Sorting Test (WSCT), an assessment tool designed more than 60 years ago that sensitively measures the ability to learn new rules on the fly and apply them to complete a task.

Electroencephalography (“brain wave”) studies of normal individuals have revealed that gamma oscillations — neural activity with a regular rhythm between 30 and 120 cycles per second — increase in the front of the brain during cognitive tasks related to the WCST, but “these gamma oscillations are blunted in individuals with schizophrenia,” said Sohal, the Staglin Family-IMHRO Assistant Professor of Psychiatry at UCSF.

Because gamma oscillations emerge from the activity of FS interneurons, and because postmortem studies of the brains of individuals with schizophrenia have shown biochemical abnormalities in FS interneurons, many researchers have concluded these neurons must play some role in the cognitive symptoms of the disorder.

In the new research, first author and postdoctoral fellow Kathleen K.A. Cho, Ph.D., led a team that made use of mice developed in the UCSF laboratory of John L.R. Rubenstein, M.D., Ph.D., the Nina Ireland Distinguished Professor in Child Psychiatry. These mice carry only one copy of two genes known as Dlx5 and Dlx6, which govern the proper assembly of FS interneuron circuitry as the brain develops. An intriguing characteristic of these mice is that FS interneurons only become abnormal at a developmental stage corresponding to human post-adolescence, which is when symptoms of schizophrenia usually begin to emerge.

In the new research when these mice performed a “rule-shift” task — designed to emulate important features of the WCST — at a young age, their performance was indistinguishable from that of normal mice. But they showed significant deficits when they performed the task as young adults.

To firmly establish that interneuron abnormalities were responsible for this declining performance, the researchers disrupted gamma oscillations in normal adult mice with a technique that allowed them to shine a light into the brain to inhibit the activity of interneurons in the prefrontal cortex, including FS interneurons. These mice performed as poorly on the rule-shift task as those lacking Dlx5 and Dlx6.

Conversely, the scientists used a similar technique to restore gamma oscillations by stimulating interneurons in the prefrontal cortex with light in mice lacking Dlx5 and Dlx6, and the mice performed the task as well as normal mice.

Finally, when the team gave low doses of clonazepam (Klonopin), which modulates the GABA neurotransmitter system employed by FS interneurons, to mice lacking Dlx5 and Dlx6, the mice again performed the rule-shifting task normally.

The cognitive improvements following direct interneuron stimulation persisted for a week after the experiments, suggesting that targeting FS interneuron dysfunction may result in durable improvements in cognitive function in schizophrenia.

Clonazepam and other GABA-modulating drugs such as lorazepam (Ativan) and diazepam (Valium) are now used to treat anxiety associated with schizophrenia, but usually at higher doses that also increase sedation, which may mask any cognition-enhancing effect. Lower doses, or better-designed compounds that specifically target FS interneurons in the prefrontal cortex, might be better options, Sohal said, adding that measuring gamma oscillations may be a useful guide in developing these approaches.

On a more speculative level, Sohal added, it may be possible to increase gamma oscillations to improve cognition using recently developed, non-invasive brain stimulation technologies such as transcranial magnetic or direct-current stimulation (TMS or tDCS), or even by combining meditation with biofeedback.

“Meditation has been shown to potently increase gamma oscillations, and you may be able to teach patients to increase gamma oscillations by themselves,” said Sohal. “Now that we know that gamma oscillations are directly related to cognitive performance, it’s certainly an interesting idea to pursue.”

In addition to Sohal, Cho and Rubenstein, researchers participating in the study included former postdoctoral fellow Renee Hoch, Ph.D.; Anthony Lee, a student in USCF’s M.D./Ph.D. program; and laboratory specialist Tosha Patel.

The work was supported by the Staglin Family and the International Mental Health Research Organization; the National Institute of Mental Health; the Alfred P. Sloan Foundation; the National Institutes of Health; and the Brain and Behavior Research Foundation.

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Scientists describe novel drug mechanism that fights brain cancer

UC Davis findings could lead to better therapies against many hard-to-treat cancers.

By Dorsey Griffith, UC Davis

Researchers at UC Davis have developed and characterized a molecule that interferes with the internal regulation of cancer cells, causing them to self-destruct. This novel mechanism was found to be effective against glioma cells – responsible for a usually fatal type of brain cancer – and could be applicable to other highly aggressive cancers.

The article, to be published in the April 2015 issue of Molecular Pharmacology, is available online at doi:10.1124/mol.114.096602.

“We have elucidated the mode of action of a drug that destroys glioma cells in a manner that has not previously been described,” said Nagarekha Pasupuleti, lead author of the study and project scientist in the Department of Neurology. “We anticipate that it will lead to new treatments to fight cancers that are resistant to standard therapies.”

The investigators performed a series of studies utilizing high-content analysis, which quantifies changes in living cells in response to a drug treatment. The lab focused on the effects of a patented small molecule previously developed at UC Davis, known as UCD38B, on four different human glioma cell lines.

Gliomas arise from glia cells in the brain, which provide structural support and protection to neurons. Treatment of glioma typically involves a combination of surgery, radiation therapy and chemotherapy. Although apparently eradicated from the body after treatment, the cancer has a high rate of recurrence.

According to Pasupuleti, the problem with conventional therapy is that a subpopulation of non-dividing cancer cells tends to remain unaffected by treatment. These cells, which have many properties in common with normal stem cells, remain quiescent for a time, later replicate and regenerate the tumor. This population of glioma-initiating cancer cells resides in tumor regions having negligible or no blood supply and minimal oxygen, making them very difficult to destroy.

The research team’s study showed that UCD38B is effective against such non-dividing glioma cells, as well as dividing cells destroyed by conventional therapy. They found that UCD38B acts by targeting a cellular regulatory system called the urokinase plasminogen activator system. This system is normally important when tissue needs to be re-organized, such as during wound healing, a process that requires new cells to be made and others destroyed. Components of the urokinase plasminogen activator system have been found to be highly active in many aggressive cancers, including gliomas, as well as metastatic breast, lung and pancreatic cancers. The system is believed to play an important role in the ability of cancer cells to grow uncontrollably and metastasize to other parts of the body.

UCD38B disrupts the intracellular components of the urokinase plasminogen activator system. After entering glioma cells, UCD38B causes “mis-trafficking” of urokinase plasminogen activator system components to the wrong region of the cancer cell, ultimately triggering the cells to signal their own destruction rather than proliferate. UCD38B does this by disrupting the cell’s endosomal transport system, which normally functions to direct cellular components to areas where they may be needed, or if not needed, destroyed. Within a few hours of administration, UCD38B causes plasminogen activator system components to be sent to mitochondria near the cell nucleus instead of the cell surface, causing factors to be released that destroy the cell.

Preliminary studies in rodents implanted with human glioma cells have found that a new small molecule based upon UCD38B is very effective in destroying this population of hypoxic glioma cells within the tumor without evidence of adverse effects. The research team will continue these studies and, in collaboration with the UC Davis School of Veterinary Medicine, hopes to try the drug in dogs with high-grade glial brain cancers, for which there are no other treatment options.

“Understanding the drug mechanism of action of UCD38B and its more potent derivatives is the culmination of many years of work of characterizing the processes causing cancer recurrence and developing molecules that target therapeutically resistant cancer cell types,” said Fredric Gorin, principal investigator, chair of the UC Davis Department of Neurology School of Medicine and professor of molecular biosciences in the UC Davis School of Veterinary Medicine. “We are hopeful that this new class of drug will one day become an important adjunct to conventional therapies in fighting these especially difficult-to-treat cancers.”

The article is titled “Mis-trafficking of endosomal urokinase proteins triggers drug-induced glioma non-apoptotic cell death.”

In addition to Gorin and Pasupuleti, Ana Cristina Grodzki of the Department of Molecular Biosciences in the UC Davis School of Veterinary Medicine, was a co-author and played an important role in quantifying the endosomal trafficking caused by UCD38B.

This research was funded by National Institutes of Health, Neurological Sciences grants (NS040489 NS060880) to the UC Davis School of Medicine, the UC Davis Research Investments in Science and Engineering (RISE) and the MIND Institute Intellectual and Developmental Disabilities Research Center (IDDRC) grant (U54 HD079125).

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Cystic fibrosis discovery may lead to new strategy to help patients breathe easier

UCSF-led team ID’s why disease thickens mucus in lungs, explores possible new treatments.

By Pete Farley, UC San Francisco

A team led by UC San Francisco professor of medicine John Fahy, M.D., has discovered why mucus in the lungs of people with cystic fibrosis (CF) is thick, sticky and difficult to cough up, leaving these patients more vulnerable to lung infection.

Fahy and his team found that in CF – contrary to previous belief – inflammation causes new molecular bonds to form within mucus, transforming it from a liquid to an elastic sludge.

The scientists also made headway in the lab in exploring a potential new therapeutic approach to dissolve those bonds and return the mucus to a liquid that is easier for the lungs to clear.

CF is a lifelong inherited disease that affects the lungs and digestive system. There is no cure. About 30,000 children and adults in the United States have CF.

Fahy said that the research, a collaborative effort between UCSF, University College Dublin (UCD) in Ireland and the Cleveland Clinic (CC) in Ohio, has implications for other lung conditions characterized by thickened mucus, such as chronic obstructive pulmonary disorder (COPD) and asthma.

The work was reported in today’s (Feb. 25) issue of Science Translational Medicine.

Polymers – naturally-occurring molecules in mucus that form long chains – are the key to the discovery.

Until now, scientists had thought that CF mucus is thicker than healthy mucus because it has a greater concentration of DNA polymers. To test that idea, Fahy and his group exposed mucus samples taken from CF patients to two current CF medications: Pulmozyme, a drug that breaks up DNA polymers, and N-acetylcysteine (NAC), which targets disulfide bonds between mucin polymers. Mucin is a protein that is the major constituent of mucus.

“We thought Pulmozyme would be more effective than NAC in liquefying the mucus, because CF sputum contains lots of DNA,” said Fahy. “But to our surprise, NAC worked much better.”

Using confocal microscopy, the scientists learned why: CF mucus consists of a dense core of mucin with a layer of DNA wrapped around it, like a thin blanket draped over a solid pillow. Thus, while Pulmozyme makes mucus less stiff by eliminating DNA, NAC succeeds in liquefying it by breaking up the mucin.

Fahy and his team then investigated why mucin in CF is so compacted. They found that mucin polymers become linked together crosswise by newly forged disulfide bonds. Fahy likened the polymers to logs floating down a river. “The logs can float down the river as long as they are floating independently,” he said. “But if you bolt them together side to side, they will clog the river.”

The researchers found that inflammation causes the extra disulfide bonds to form, when mucin polymers are exposed to highly reactive oxygen molecules released by inflammatory cells in a process called oxidative stress.

This observation was confirmed by a device invented by lead investigator Leo Shaopeng Yuan, of the UCSF Cardiovascular Research Institute. In separate chambers, mucus from healthy volunteers was exposed to pure oxygen and pure nitrogen. The mucus exposed to oxygen became thick and elastic within seconds. The mucus exposed to nitrogen remained liquid.

“This qualitative change, driven by oxidation, happens with other natural polymers,” said Fahy. “Think of latex, which starts out as liquid tree sap. When it’s vulcanized – a process of chemical cross-linking – it turns into the solid rubber we use in tires.”

Fahy noted that patients who are treated with pure oxygen in hospital intensive care units have long been known to develop sticky mucus. “This could be a function of the oxygen that’s used to treat them,” he said.

Finally, the research team turned its attention to the possibility of creating new treatments for CF that would target disulfide bonds in mucin polymers directly and efficiently.

NAC, which targets mucin polymer bonds, is already an approved medication used to break up mucus. “However,” said Fahy, “there are problems with it. It’s a relatively weak drug, and it smells like rotten eggs.”

Team member Stefan Oscarson, Ph.D., a medicinal chemist from UCD, designed TDG, an experimental compound that targets disulfide bonds. TDG liquefied mucus samples from CF patients much more efficiently than NAC.

Fahy cautioned that TDG cannot yet be given to human beings. He noted that while the team has applied for funding to develop their promising new therapeutic approach, “there are at least five years of testing ahead before we can say we have a new medication.”

Fahy predicted that the new finding will explain the reason for thick mucus in other lung diseases known to be associated with oxidative stress, including COPD and asthma. “We’re very confident that we’ve uncovered a ubiquitous mechanism here,” he said.

Co-authors of the study are Martin Hollinger, Ph.D., of UCD; Marrah E. Lachowicz-Scroggins, Ph.D., Sheena C. Kerr, Ph.D., Eleanor M. Duncan, M.D., and Brian M. Daniel, R.R.T., of UCSF; Sudakshina Ghosh Ph.D., Serpel C. Erzurum, M.D., Belinda Willard, Ph.D., and Stanley L. Hazen, M.D., Ph.D., of the Cleveland Clinic; Xiaozhu Huang, M.D., of UCSF; and Stephen D. Carrington, Ph.D., of UCD.

The study was supported by funds from the National Institutes of Health and Genentech.

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Technology optimizes cancer therapy with nanomedicine drug combinations

UCLA bioengineers develop platform that offers personalized approach to treatment.

By Brianna Aldrich, UCLA

In greater than 90 percent of cases in which treatment for metastatic cancer fails, the reason is that the cancer is resistant to the drugs being used. To treat drug-resistant tumors, doctors typically use multiple drugs simultaneously, a practice called combination therapy. And one of their greatest challenges is determining which ratio and combination — from the large number of medications available — is best for each individual patient.

Dr. Dean Ho, a professor of oral biology and medicine at the UCLA School of Dentistry, and Dr. Chih-Ming Ho, a professor of mechanical engineering at the UCLA Henry Samueli School of Engineering and Applied Science, have developed a revolutionary approach that brings together traditional drugs and nanotechnology-enhanced medications to create safer and more effective treatments. Their results are published in the peer-reviewed journal ACS Nano.

Chih-Ming Ho, the paper’s co-corresponding author, and his team have developed a powerful new tool to address drug resistance and dosing challenges in cancer patients. The tool, Feedback System Control.II, or FSC.II, considers drug efficacy tests and analyzes the physical traits of cells and other biological systems to create personalized “maps” that show the most effective and safest drug-dose combinations.

Currently, doctors use people’s genetic information to identify the best possible combination therapies, which can make treatment difficult or impossible when the genes in the cancer cells mutate. The new technique does not rely on genetic information, which makes it possible to quickly modify treatments when mutations arise: the drug that no longer functions can be replaced, and FSC.II can immediately recommend a new combination.

“Drug combinations are conventionally designed using dose escalation,” said Dean Ho, a co-corresponding author of the study and the co-director of the Jane and Jerry Weintraub Center for Reconstructive Biotechnology at the School of Dentistry. “Until now, there hasn’t been a systematic way to even know where the optimal drug combination could be found, and the possible drug-dose combinations are nearly infinite. FSC.II circumvents all of these issues and identifies the best treatment strategy.”

The researchers demonstrated that combinations identified by FSC.II could treat multiple lines of breast cancer that had varying levels of drug resistance. They evaluated the commonly used cancer drugs doxorubicin, mitoxantrone, bleomycin and paclitaxel, all of which can be rendered ineffective when cancer cells eject them before they have had a chance to function.

The researchers also studied the use of nanodiamonds to make combination treatments even more effective. Nanodiamonds — byproducts of conventional mining and refining operations — have versatile characteristics that allow drugs to be tightly bound to their surface, making it much harder for cancer cells to eliminate them and allowing toxic drugs to be administered over a longer period of time.

The use of nanodiamonds to treat cancer was pioneered by Dean Ho, a professor of bioengineering and member of the UCLA Jonsson Comprehensive Cancer Center and the California NanoSystems Institute.

“This study has the capacity to turn drug development, nano or non-nano, upside-down,” he said. “Even though FSC.II now enables us to rapidly identify optimized drug combinations, it’s not just about the speed of discovering new combinations. It’s the systematic way that we can control and optimize different therapeutic outcomes to design the most effective medicines possible.”

The study found that FSC.II-optimized drug combinations that used nanodiamonds were safer and more effective than optimized drug-only combinations. Optimized nanodrug combinations also outperformed randomly designed nanodrug combinations.

“This optimized nanodrug combination approach can be used for virtually every type of disease model and is certainly not limited to cancer,” said Chih-Ming Ho, who also holds UCLA’s Ben Rich Lockheed Martin Advanced Aerospace Tech Endowed Chair. “Additionally, this study shows that we can design optimized combinations for virtually every type of drug and any type of nanotherapy.”

Both Dean Ho and Chih-Ming Ho have collaborated with other researchers and have validated FSC.II’s efficacy in many other types of cancers, infectious diseases and other diseases.

Other co-authors were Hann Wang, Dong-Keun Lee, Kai-Yu Chen and Kangyi Zhang, all of UCLA’s department of bioengineering, School of Dentistry, California NanoSystems Institute and Jonsson Cancer Center; Jing-Yao Chen of UCLA’s department of chemical and biomolecular engineering; and Aleidy Silva of UCLA’s department of mechanical and aerospace engineering.

The work was supported in part by the National Cancer Institute, the National Science Foundation, the V Foundation for Cancer Research, the Wallace H. Coulter Foundation, the Society for Laboratory Automation and Screening, and Beckman Coulter Life Sciences.

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Doctorate program will study substance abuse, its consequences

Collaboration between UC San Diego, SDSU among first in nation.

By Scott LaFee, UC San Diego

A new Joint Doctoral Program (JDP) in Interdisciplinary Research on Substance Use has been launched by the Division of Global Public Health in the UC San Diego School of Medicine and San Diego State University’s School of Social Work. The program will emphasize research devoted to studying the use and misuse of alcohol and drugs – and related social and health consequences.

“This program is the first of its kind,” said JDP co-director Steffanie Strathdee, Ph.D., professor and head of the UC San Diego Global Health Initiative. “Given that substance use has a growing health and societal impact in the U.S. and globally, this program could not come at a better time.”

The JDP will focus on research designed to identify and assess substance use risk and create intervention programs for preventing or ameliorating high‐risk behaviors related to substance use. It will include training to craft and evaluate disease prevention and health promotion recommendations and help guide public health policies.

María Luisa Zúñiga, Ph.D., JDP co-director and associate professor in SDSU’s School of Social Work, said “SDSU and UC San Diego have a long history of jointly offering cutting edge, high-demand programs. This new doctoral program is designed to train the next generation of researchers to lead interdisciplinary research efforts that will meaningfully address substance use issues of national and global impact. Our graduates will be highly sought after in fields including medicine, social work and public health, as well as research firms and governmental health departments.”

The new JDP is the 14th such program offered by UC San Diego and SDSU. Others include highly acclaimed programs in public health and clinical psychology.

Funding from SDSU Division of Academic Affairs and College of Health and Human Services will cover tuition fees and a teaching associate stipend for four students per year for up to four years. Students will spend the first year of study at SDSU, the second at UC San Diego and subsequent years working with faculty from both campuses.

For more information on the joint doctoral program in Interdisciplinary Substance Use Studies, visit socialwork.sdsu.edu/degrees-programs/graduate-programs/phd-substance-use-studies/phd-overview.

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Scientists limit accelerated cellular aging caused by meth use

Grasp of underlying molecular mechanisms could improve addiction recovery efforts.

Daniele Piomelli, UC Irvine

The ravaged faces of methamphetamine addicts tell a terrible tale – abusing the drug dramatically accelerates aging. Now scientists from UC Irvine and the Italian Institute of Technology have discovered how this occurs at the cellular level and identified methods to limit the process.

With funding from the National Institute on Drug Abuse to probe the effects of methamphetamine on the “lipidome” (the complete lipid profile of representative organs and tissues) in rats that self-administer the drug, UCI’s Daniele Piomelli and his IIT colleagues found that its use caused abnormalities in cellular fat metabolism, triggering extreme inflammation marked by a considerable rise in the formation of ceramides, pro-inflammatory molecules that can foster cell aging and death.

Study results appear in the open-access online journal PLOS ONE.

Methamphetamine is a highly addictive psychostimulant that profoundly damages the brain and other body organs. Postmortem examinations of human tissues have linked use of the drug to diseases associated with aging, such as coronary atherosclerosis and pulmonary fibrosis, but the molecular mechanisms underlying these findings have remained unknown.

The Piomelli team learned that this cellular cascade involves the recruitment of nuclear factor kappa beta, a protein that under healthy conditions helps control DNA encoding of proteins. But as the cell is flooded with methamphetamine-induced signaling, nuclear factor kappa beta triggers excessive signaling in pathways that engender dramatic increases in ceramide activity.

“We found this signaling process to be key for advanced cellular aging, which is at the heart of the accelerated aging influenced by methamphetamine abuse,” said Piomelli, the Louise Turner Arnold Chair in the Neurosciences.

Having identified these mechanisms, the researchers tested existing inhibitors of nuclear factor kappa beta signaling, which succeeded in limiting ceramide formation. This prevented methamphetamine-induced cell aging and systemic inflammation in rats self-administering the drug, curtailing their health deterioration.

“These results suggest new therapeutic strategies to reduce the adverse consequences of methamphetamine abuse and improve the effectiveness of abstinence treatments,” Piomelli said.

He is currently working with colleagues at the Italian Institute of Technology, in Genoa, to create a pharmaceutical application of these inhibitor compounds.

Giuseppe Astarita and Agnesa Avanesian-Thomas of UCI; Benedetto Grimaldi, Natalia Realini and Abdul Basit of the Istituto Italiano di Tecnologia; and Zuzana Justinova, Leigh V. Panlilio and Steven R. Goldberg of the National Institute on Drug Abuse contributed to the study – supported by NIDA through grant RC2 DA028902 and its intramural research program.

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