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Melanie Ott Presented with DiNA Award 2017

Congratulations, Melanie for being honored for your work with the Promoting Underrepresented Minorities Advancing in the Sciences (PUMAS) internship program by the California Life Sciences Association (CLSA). PUMAS supports educational activities that enhance diversity in biomedical research. Established in 2014 by Melanie Ott and Kathy Ivey at the Gladstone Institutes, the program encourages students from historically underrepresented backgrounds, who are currently attending a community college, to pursue undergraduate and graduate degrees in science, technology, engineering, or mathematics (STEM).

Each summer, qualified students are matched with a scientific mentor to gain hands-on biomedical research experience in a Gladstone laboratory. To date, 24 interns have gone through the PUMAS program, and three have since returned to Gladstone as research associates.

The PUMAS program is funded by the National Heart, Lung, and Blood Institute of the National Institutes of Health.

Gladstone is an independent, nonprofit life science research organization that uses visionary science and technology to overcome disease. To ensure their work does the greatest good, Gladstone researchers focus on conditions with profound medical, economic, and social impact—unsolved diseases. Gladstone has an academic affiliation with the University of California, San Francisco.

Cancer Drug Can Reactivate HIV

SAN FRANCISCO, CA—August 24, 2017

People living with HIV must take a combination of three or more different drugs every day for the rest of their lives. Unfortunately, by following this strict treatment plan, they can suffer from side effects ranging from mild dizziness to life-threatening liver damage. However, if they stop taking the drugs, the virus hiding inside their cells can spontaneously resurface.

In fact, the latent HIV, which can hide in cells for many years, is a critical barrier to a cure. Researchers are exploring two main strategies to tackle this problem––reactivate and destroy the latent virus (called “shock and kill”) or find a way to silence it for good.

In an effort to tackle both strategies, a team of scientists at the Gladstone Institutes studies drugs that disrupt latency and could eventually be used to treat infected patients. They recently discovered how a new drug called JQ1, which is currently in early-phase human cancer trials, can reactivate latent HIV.

The Key Is the Short Form
“Our discovery was born out of frustration,” explained Gladstone Senior Investigator Melanie Ott, whose study was published today in the journal Molecular Cell. “We already knew that the drug JQ1 targets a protein called BRD4, but our experiments were not yielding consistent results. Then, we started looking at different forms of the protein and, unexpectedly, found that a short form was the key to silencing HIV.”

By identifying this new role for the short form of BRD4, Ott’s team could finally explain a mechanism that controls HIV latency. They showed that the drug JQ1 targets and removes the short form of BRD4, which then allows the virus to make copies of itself.

“Many people in the field don’t even know that a short form of BRD4 exists,” said Ryan Conrad, a postdoctoral scholar in Ott’s lab and first author of the study. “While uncovering the role of this protein in HIV, we discovered that it may also be involved in fighting other viruses related to HIV. Therefore, our findings could provide new insights into an ‘old’ cellular defense mechanism against invading viruses.”

The study could also impact a broader range of diseases, given that the drug JQ1 is already being tested as a way to target the BRD4 protein to treat cancer, heart failure, and inflammation.

A Holistic Approach to Curing HIV
Many scientists concentrate on the “shock and kill” strategy as a way to cure HIV, but more and more of them are shifting their focus to silencing the virus. The mechanism discovered at Gladstone can support both strategies—manipulating the BRD4 protein either to help HIV resurface or to strengthen the body’s capacity to suppress it.

“Silencing and reactivating HIV are often seen as competing approaches, but I think they could actually be combined to develop more effective therapies in the future,” added Ott, who is also a professor in the Department of Medicine at the University of California, San Francisco (UCSF). “You could start by shocking and killing the virus that’s easy to target, then use silencing mechanisms to slow the resurfacing of latent virus.”

This strategy could potentially allow patients to stop taking drugs, and for several years to elapse before the virus reactivates. By that time, the immune system could be strong enough to eliminate the virus as it surfaces.

“That’s how I see the future of HIV cure research,” said Ott.

Scientists may not completely eliminate HIV overnight, but Ott’s team is working to find a way to target the virus so people who are infected can stop continuously taking pills.

Publication: Conrad RJ, Fozouni P, Thomas S, Sy H, Zhang Q, Zhou MM, Ott M. The Short Isoform of BRD4 Promotes HIV-1 Latency by Engaging Repressive SWI/SNF Chromatin-Remodeling Complexes. Mol Cell. 2017 Aug 17. pii: S1097-2765(17)30549-X. doi: 10.1016/j.molcel.2017.07.025.

News and Highlights Community News Research News Media Coverage Videos For Journalists Study Reveals a New Method to Address a Major Barrier to Eradicating HIV

Scientists at the Gladstone Institutes discovered that an enzyme called SMYD2 could be a new therapeutic target for flushing out the HIV that hides in infected individuals. Overcoming this latent virus remains the most significant obstacle to a cure.

While drug therapy allows people living with HIV to lead a relatively normal life, it also comes with adverse effects. In addition, patients must stay on the drugs for life to prevent the virus hiding in their body from reactivating. In the early stages of infection, HIV hides in viral reservoirs in a type of immune cells called T cells. This dormant, or latent, virus can then spontaneously reactivate and rekindle infection if drug therapy is stopped.

To eliminate HIV latency, scientists are exploring a “shock and kill” strategy that would use a combination of drugs to wake up the dormant virus, then act with the body’s own immune system to eliminate the virus and kill infected cells. Previous research has had limited success in efficiently reactivating latent HIV, so scientists are working to find new, more effective drugs.

“Our study focused on a class of enzymes called methyltransferases, which have emerged as key regulators of HIV latency,” explained Melanie Ott, MD, PhD, a senior investigator at Gladstone and lead author of the study published in the scientific journal Cell Host & Microbe. “These enzymes have also become increasingly important in disease development, particularly cancer, and efforts have intensified to develop specific pharmacological inhibitors targeting them.”

“We systematically screened over 50 methyltransferases to determine which ones regulate latency in infected T cells,” said Daniela Boehm, postdoctoral scholar in the Ott laboratory and first author of the study. “We identified SMYD2 as a regulating enzyme, and found that inhibiting it reactivates, or wakes up, latent cells. SMYD2 could therefore be used as a therapeutic target in the shock and kill strategy.”

Although SMYD2 was not previously considered a target for HIV, pharmacological inhibitors are already being developed against this enzyme due to its effect on various cancer tumors.

“Our findings offer new biological and mechanistic insights into how latency functions,” added Ott, who is also a professor in the Department of Medicine at the University of California, San Francisco (UCSF). “They also suggest potential translational applications. Through a valuable collaboration with our industry partners, we obtained samples of small molecules in pre-clinical development that target SMYD2 and could potentially activate latent HIV.”

In collaboration with Warner C. Greene, MD, PhD, the researchers tested the small molecules that inhibit SMYD2 in human cells.

“We found that these small SMYD2 inhibitors were able to activate the virus in latently infected T cells isolated from HIV patients,” said Greene, senior investigator and director of the Gladstone Institute of Virology and Immunology.

“Our findings provide the basis for a new model of HIV latency wherein SMYD2 contributes to durably repressing the latent virus,” said Ott. “They also underscore the emerging ties between cancer and HIV treatment through shared pharmacological targets. Though we are still far from a human application, it is exciting to know that data from this study might readily connect with clinical efforts.”

Publication: Boehm D, Ott M. Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines. Bio Protoc. 2017 May 20;7(10). pii: e2290. doi: 10.21769/BioProtoc.2290.

Using Viruses to Find the Cellular Achilles Heel

SAN FRANCISCO, CA—January 22, 2015­—A study from researchers at the Gladstone Institutes has exposed new battle tactics employed by the hepatitis C virus (HCV). Published in the January 22 issue of Molecular Cell, the investigators created full protein interaction maps—interactomes—of where the virus comes into contact with the host proteins during the course of infection. Through these protein interactions, the scientists not only gained insight into the virus, they also uncovered a common set of host proteins that are targeted by various infections. Their results suggest that these proteins and the cellular processes they govern are the most crucial—in effect, the collective Achilles heel—for both the human body and its viral invaders.

“Viruses are fantastic tools for shedding new light on human biology,” says Nevan Krogan, PhD, a senior investigator at the Gladstone Institutes and a corresponding author. “Viruses are relatively simple organisms—often they only have about 10-20 genes—but they wreak havoc on our system by targeting key proteins and essential functional pathways in every major biological process. This gives us great insight into what the critical mechanisms are that are being hijacked during infection, and it helps us to develop new strategies for preventing or stemming disease.”

Dr. Krogan, who is also a professor of cellular and molecular pharmacology at the University of California, San Francisco (UCSF) and director of the UCSF branch of QB3, partnered with Gladstone senior investigator Melanie Ott, MD, PhD, to map the interaction between the proteins in HCV and those in the human liver cells that HCV infects. This resulted in over 5,000 virus-host protein-to-protein interactions, which the investigators narrowed down to 139 key connections that are necessary for HCV infection, involving all 10 HCV proteins and 133 proteins in the host liver cells.

Although patients have benefited from numerous advancements in the treatment of HCV, how the virus damages the liver remains unknown. The HCV interactome map, led by first authors Holly Ramage, PhD, and G. Renuka Kumar, PhD, may help on this front.

“There’s a lot we still don’t know about HCV, like how it infects the cell, what processes it disrupts, and why it’s so harmful to the body,” explains Dr. Ott, who is also a professor of medicine at UCSF. “The protein interactome offers us an unbiased and global view of how the virus is affecting the infected cells, and this can help us to start answering a lot of the important questions. Ultimately, this information may direct us to new leads for preventative treatments for associated liver pathologies, like fibrosis and cancer.”

By removing the interacting host proteins from the cell one at a time, the researchers were able to determine what their functional contribution was in the infection process: whether the host proteins were hijacked by the virus and used to spread infection, or whether they were part of a defense mechanism against the virus. This revealed a new critical host mechanism that HCV inactivates and usurps to support its own survival and replication.

Publication: Ramage HR, Kumar GR, Verschueren E, Johnson JR, Dollen Von J, Johnson T, Newton B, Shah P, Horner J, Krogan NJ, Ott M (2015) A combined proteomics/genomics approach links hepatitis C virus infection with nonsense-mediated mRNA decay. Mol Cell 57:329–340.

Melanie Receives 2014 Avant-Garde Award for HIV/AIDS Research

Three scientists, including Gladstone’s Melanie Ott, MD, PhD, have been chosen to receive the 2014 Avant-Garde Award for HIV/AIDS Research from the National Institute on Drug Abuse (NIDA), part of the National Institutes of Health. The three scientists will each receive $500,000 per year for five years to support their research.

Project: A new model of accelerated immune aging in HIV-infected drug users

Dr. Ott will investigate the role of an enzyme (SIRT-1) in slowing accelerated immune aging resulting from either long-term HIV infection or regular drug use. Because SIRT-1 appears to protect against overworked immune activation that can eventually exhaust immune cell functions, new therapies aimed at this enzyme could delay immune aging and its related health risks in HIV-infected drug users.

“The goal of our research is to transform our understanding of how HIV and drug abuse affect the immune system and the aging process,” Ott said. “We hope to identify novel links between HIV, abused substances and the biological pathways of aging that lead to potential therapeutic strategies to slow the accelerated immune aging in this patient population.”

“These innovative approaches can shed light on mechanisms through which HIV damages or circumvents the immune system, and how these effects interact with those of drugs of abuse” said NIDA Director Nora D. Volkow, M.D. “By learning more about these underlying processes, not only might this research slow the progression and transmission of HIV infection, but it could improve the long-term health of those already infected.”

Navigating the Road from DNA to RNA—and Beyond

November 7, 2013—One of biology’s most fundamental processes is something called transcription. It is just one step of many required to build proteins—and without it life would not exist. However, many aspects of transcription remain shrouded in mystery. But now, scientists at the Gladstone Institutes are shedding light on key aspects of transcription, and in so doing are coming even closer to understanding the importance of this process in the growth and development of cells—as well as what happens when this process goes awry.

In the latest issue of Molecular Cell, researchers in the laboratory of Gladstone Investigator Melanie Ott, MD, PhD, describe the intriguing behavior of a protein called RNA polymerase II (RNAPII). The RNAPII protein is an enzyme, a catalyst that guides the transcription process by copying DNA into RNA, which forms a disposable blueprint for making proteins. Scientists have long known that RNAPII appears to stall or “pause” at specific genes early in transcription. But they were not sure as why.

“This so-called ‘polymerase pausing’ occurs when RNAPII literally stops soon after beginning transcription for a short period before starting up again,” explained Dr. Ott. “All we knew was that this behavior was important for the precise transcription of DNA into RNA, so we set out to understand how, when and—most importantly—why.”

The research team focused their efforts on a segment of RNAPII called the C-terminal domain, or CTD. This section is most intimately involved with transcription regulation. Previous research had found that CTD’s chemical structure is modified before and during transcription. However, the combinations of modifications as well as precisely how they influence or control transcription remained unclear. So in laboratory experiments on cells extracted from mammals, the researchers took a closer look.

The first breakthrough came when the research team identified a new type of modification, known asacetylation, which regulated transcription.

“Our next breakthrough occurred when we pinpointed the precise locations on the CTD where acetylation occurred—and realized it was unique to higher eukaryotes,” explained Sebastian Schröder, PhD, the paper’s first author. “We then wanted to see how this mammalian-specific acetylation fit into the realm of polymerase pausing.”

Now that the team knew where the CTD became acetylated, their next goal was to find out when. Clues to the timing of acetylation came in experiments where they mutated RNAPII so that CDT was unable to become acetylated. In these cases, the length of polymerase pausing dropped, and the necessary steps for the completion of transcription failed to occur. Additional experiments revealed the elusive timeline of acetylation and transcription.

“RNAPII binds to DNA to prepare for transcription. Shortly after that we see polymerase pausing—at which point the CTD becomes highly acetylated,” continued Dr. Shröder. “Soon after the pause, CTD is thendeacetylated—the original modification is reversed—and transcription continues without a hitch.”

Polymerase pausing is not unique to mammals—in fact it was characterized in HIV, the virus that causes AIDS, many years ago—but the fact that the CTD becomes acetylated just before or during the time when transcription is paused appears to be unique. Drs. Ott and Schröder argue that CTD acetylation is a stabilizer, preparing RNAPII for efficient completion of transcription and slowing down the process to make sure everything is functioning correctly—not unlike the final ‘systems check’ a pilot must perform before takeoff.

These findings offer important insight into the relationship between acetylation and transcription. And given the importance of transcription in the growth and maturation of cells in general, the team’s result stands to inform scientists about a variety of cellular processes. These include, for example, the mechanisms behind stem-cell development and what happens when normal cellular growth spirals out of control, such as in cancer.

“However, there is still much we don’t know about acetylation as it relates to transcription,” said Dr. Ott. “For example, if CTD acetylation is important for stabilizing transcriptional pausing, why do we also see CTD acetylation at non-paused genes, although at different locations? Further, we believe there may be other steps in the transcription cycle that depend upon acetylation. Our most immediate goal is to find them. By doing so, we hope to deepen our understanding of one of nature’s most elegant biological processes.”

Publication: Schröder S, Herker E, Itzen F, He D, Thomas S, Gilchrist DA, Kaehlcke K, Cho S, Pollard KS, Capra JA, Schnolzer M, Cole PA, Geyer M, Bruneau BG, Adelman K, Ott M (2013) Acetylation of RNA Polymerase II Regulates Growth-Factor-Induced Gene Transcription in Mammalian Cells. Mol Cell 52:314–324.

Gladstone Scientists Link Hepatitis C Virus Infection to Fat Enzyme in Liver Cells

SAN FRANCISCO, CA—October 10, 2010—Scientists at the Gladstone Institute of Virology and Immunology (GIVI) have found that an enzyme associated with the storage of fat in the liver is required for the infectious activity of the hepatitis C virus (HCV). This discovery may offer a new strategy for treating the infection.

More than 160 million people are infected throughout the world, and no vaccine is available to prevent further spread of the disease. Current treatments are not effective against the most common strains in the US and Europe. The study, published in the journal Nature Medicine, shows that the enzyme DGAT1 is a key factor in HCV infection. With several potential DGAT1 inhibitors already in the drug-development pipeline, a treatment for HCV may be possible in the near future.

“Our results reveal a potential ‘Achilles heel’ for HCV infection,” said Melanie Ott, MD, PhD, senior author on the study. “Several DGAT1 inhibitors are already in early clinical trials to treat obesity-associated diseases. They might also work against HCV.”

At first glance, the HCV lifecycle is fairly simple. The virus enters the cell. One large protein is produced and cut into several smaller viral enzymes and proteins that build the virus. The RNA genome is copied, and the new RNAs and structural proteins are used to make new virus particles that are released into the blood stream to infect more cells. These processes were thought to occur at specialized membranes inside the cell. However, recently it has been shown that fat droplets are critically involved.

Fat droplets, which store fat in cells, have become a hot new topic in biology. DGAT1 is one of the enzymes that help to form fat droplets. The Gladstone team, led by Eva Herker, PhD, discovered that HCV infection and viral particle production are severely impaired in liver cells that lack DGAT1 activity.

“DGAT enzymes produce the fat that is stored in the droplets that are important for HCV replication, so we wondered if inhibiting those enzymes might disrupt the viral life cycle,” said Dr. Herker. “We found that HCV specifically relies on one DGAT enzyme, DGAT1. When we inhibit DGAT1 with a drug, the liver still produces fat droplets through another DGAT enzyme but these droplets cannot be used by HCV.”

The team sought to identify which step in the HCV lifecycle requires DGAT1. They found that DGAT1 interacts with one viral protein, the viral nucleocapsid core protein, required for viral particle assembly. The core protein normally associates with the surface of fat droplets but cannot do so when DGAT1 is inhibited or missing in infected cells.

Researchers at Gladstone Institute of Cardiovascular Disease had previously cloned DGAT1.

Charles Harris, Robert V. Farese, Jr. and Katrin Kaehlcke were part of the Gladstone team. Celine Hernadez, Arnaud Charpentier and Arielle Rosenberg supported the research from the Universite Paris Descartes.

This work was supported by funds from the Gladstone Institutes, the Hellman Family Foundation, the US National Institutes of Health and the UCSF Liver Center. Additional support was provided through fellowships from the Human Frontiers Science Program, the Agence nationale de recherches sur le sida et les hepatites virales, and a training grant from the National Institute of Diabetes and Digestive and Kidney Diseases.

Publication: Herker E, Harris C, Hernandez C, Carpentier A, Kaehlcke K, Rosenberg AR, Farese RV, Ott M (2010) Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1. Nat Med 16:1295–1298.