2-Drug Combination May Boost Immunotherapy Responses in Lung Cancer Patients

Johns Hopkins Kimmel Cancer Center researchers and colleagues have identified a novel drug combination therapy that could prime nonsmall cell lung cancers to respond better to immunotherapy. These so-called epigenetic therapy drugs, used together, achieved robust anti-tumor responses in human cancer cell lines and mice.

During the study, published Nov. 30, 2017, in the journal Cell, a team of researchers led by graduate student Michael Topper; research associate Michelle Vaz, Ph.D.; and senior author Stephen B. Baylin, M.D., combined a demethylating drug called 5-azacytidine that chemically reignites some cancer suppressor genes’ ability to operate, with one of three histone deacetylase inhibitor drugs (HDACis). The HDACis work against proteins called histone deacetylases that are involved in processes, such as cell copying and division, and can contribute to cancer development. The combination therapy triggered a chemical cascade that increased the attraction of immune cells to fight tumors and diminished the work of the cancer gene MYC. Based on these findings, investigators have launched a clinical trial of the combination therapy in patients with advanced, nonsmall cell lung cancer.

The development of therapeutic approaches for patients with lung cancer has been a critical medical need, says Baylin, the Virginia and Daniel K. Ludwig Professor of Cancer Research at the Kimmel Cancer Center. While immune checkpoint therapy has been “a tremendous step forward, less than half of patients with lung cancer have benefited to date,” he says.

“In our study, the two-drug epigenetic therapy combination worked exceedingly well, even before putting in the immune checkpoint inhibitors,” Baylin says. “In animal models of lung cancer, the two agents either prevented cancer from emerging or blunted the effects of more aggressive cancers. In both scenarios, a large component of the results involved an increase in immune recognition of the tumors.”

In a series of experiments, researchers studied the combination of 5-azacytidine with the HDACis entinostat, mocetinostat or givinostat in human cancer cell lines and in mouse models of nonsmall cell lung cancers. The treatments were found to alter the tumor microenvironment. In cancer cell lines, 5-azacytidine worked against the cancer gene MYC, causing down regulation of the entire MYC signaling program. Adding the HDACis further depleted MYC, and together the drugs subsequently caused actions that prevented cancer cell proliferation, simultaneously attracted more immune system T cells to the area of the tumor and activated these cells for tumor recognition.

In mouse models, the strongest response was observed when using 5-azacytidine plus givinostat. In one mouse model with a mutant form of nonsmall cell lung cancer, this drug combination given for three months yielded prevention of benign, precursor tumors from becoming cancers and caused 60 percent reduction of overall area of benign tumor appearance in the lungs. By contrast, a group of mice with the same form of lung cancer that were given a mock treatment universally developed large, cancerous lesions in the lungs.

In a second model of mice with established, aggressive, nonsmall cell lung cancer, treatment with an alternating schedule of 5-azacytidine with givinostat and of 5-azacytidine with mocetinostat not only reduced the growth of established, rapidly growing primary tumors but also dramatically reduced metastatic occurrence.

Baylin and colleagues at Memorial Sloan Kettering Cancer Center in New York and Fox Chase Cancer Center in Philadelphia have started a phase I/Ib clinical trial to evaluate if giving mocetinostat with a 5-azacytidinelike drug called guadecitabine can boost immune checkpoint therapy responses in patients with advanced, nonsmall cell lung cancers. The trial is part of the Van Andel Research Institute–Stand Up To Cancer Epigenetics Dream Team and is funded by Merck through the Stand Up To Cancer  (SU2C) Catalyst program, an initiative led by SU2C to bring innovative cancer treatments to patients quickly. Matthew Hellmann, M.D., an author on the paper, will lead this trial at Memorial Sloan Kettering, and Jarushka Naidoo, M.B.B.Ch., assistant professor of oncology, will lead at Johns Hopkins. For more information, click here.

Deadly Lung Cancers Are Driven by Multiple Genetic Changes

Blood-Based Cancer Tests Reveal Complex Genomic Landscape of Non-Small Cell Lung Cancers

A new UC San Francisco–led study challenges the dogma in oncology that most cancers are caused by one dominant “driver” mutation that can be treated in isolation with a single targeted drug. Instead, the new research finds one of the world’s most deadly forms of lung cancer is driven by changes in multiple different genes, which appear to work together to drive cancer progression and to allow tumors to evade targeted therapy.

These findings — published online on November 6, 2017 in Nature Genetics — strongly suggest that new first-line combination therapies are needed that can treat the full array of mutations contributing to a patient’s cancer and prevent drug resistance from arising.

“Currently we treat patients as if different oncogene mutations are mutually exclusive. If you have an EGFR mutation we treat you with one class of drugs, and if you have a KRAS mutation we pick a different class of drugs. Now we see such mutations regularly coexist, and so we need to adapt our approach to treatment,” said Trever Bivona, MD, PhD, a UCSF Medical Center oncologist, associate professor in hematology and oncology, and member of the Helen Diller Family Comprehensive Cancer Center at UCSF.

Lung cancer is by far the leading cause of cancer death worldwide. Efforts to identify the genetic mutations that drive the disease have led to targeted treatments that improve life expectancy for many patients, but these drugs produce temporary remission at best — sooner or later, cancers inevitably develop drug resistance and return, deadlier than ever.

The new UCSF-led study — which analyzed tumor DNA from more than 2,000 patients in collaboration with Redwood City–based Guardant Health — is the first to extensively profile the genetic landscape of advanced-stage non–small cell (NSC) lung cancer, the most common form of the disease.

“The field has been so focused on treating the ‘driver’ mutation controlling a tumor’s growth that many assumed that drug-resistance had to evolve from new mutations in that same oncogene. Now we see that there are many different genetic routes a tumor can take to develop resistance to treatment,” said Bivona, who is also co-director of a new UCSF-Stanford Cancer Drug Resistance and Sensitivity Center funded by the National Cancer Institute. “This could also explain why many tumors are already drug-resistant when treatment is first applied.”

New combination of anti-obesity drugs may have beneficial effects

Research conducted in the Perelman School of Medicine at the University of Pennsylvania has revealed that a unique combination of hormone-based drugs can produce enhanced weight loss in laboratory tests with obese animals. The research is to be presented this week at the Annual Meeting of the Society for the Study of Ingestive Behavior (SSIB), the foremost society for research into all aspects of eating and drinking behavior.

“Imagine a drug regimen where an obese person would cycle between different drug therapies over the course of a month to achieve a greater degree of body weight loss compared to the effects achieved with either a single drug or the continuous combination of drugs,” said senior author Dr. Matthew Hayes. His team studied the combination of two different drug classes that target different hormones: amylin and glucagon-like peptide-1 (GLP-1). They found that combined treatments acted synergistically to suppress feeding and body weight. They also discovered that the weight loss effects of chronic amylin- and GLP-1-based combination therapies could be enhanced when obese lab animals are cycled through their drug treatments. “The idea of drug-cycling is nothing new,” says lead author Kieran Koch-Laskowski. “Millions of women on birth control pills, for example, already take daily pills that cycle between drug and placebo throughout the month,” she goes on to say.

Perhaps the most exciting finding of the current data coming out of Penn is the fact that the research finds these enhanced weight loss effects with a combination of drugs that are either already FDA approved or in clinical trials for metabolic diseases, “making the translational impact of our work extremely timely and highly clinically relevant!” says Hayes. The authors are now finalizing their research to demonstrate mechanically how these two hormonal systems interact to achieve greater weight loss in the hopes of fast-tracking their findings to new clinical treatments for obesity.

A New Way to Nip AIDS in the Bud

When new AIDS virus particles bud from an infected cell, an enzyme named protease activates to help the viruses mature and infect more cells. That’s why modern AIDS drugs control the disease by inhibiting protease.

Now, University of Utah researchers found a way to turn protease into a double-edged sword: They showed that if they delay the budding of new HIV particles, protease itself will destroy the virus instead of helping it spread. They say that might lead, in about a decade, to new kinds of AIDS drugs with fewer side effects.

“We could use the power of the protease itself to destroy the virus,” says virologist Saveez Saffarian, an associate professor of physics and astronomy at the University of Utah and senior author of the study released today by PLOS Pathogens, an online journal published by the Public Library of Science.

So-called cocktails or mixtures of protease inhibitors emerged in the 1990s and turned acquired immune deficiency syndrome into a chronic, manageable disease for people who can afford the medicines. But side effects include fat redistribution in the body, diarrhea, nausea, rash, stomach pain, liver toxicity, headache, diabetes and fever.

“They have secondary effects that hurt patients,” says Mourad Bendjennat, a research assistant professor of physics and astronomy and the study’s first author. “And the virus becomes resistant to the inhibitors. That’s why they use cocktails.”

Bendjennat adds that by discovering the molecular mechanism in which protease interacts with HIV, “we are developing a new approach that we believe may be very efficient in treating the spread of HIV.”

However, he and Saffarian emphasize the research is basic, and that it will be a decade before more research might develop the approach into news AIDS treatments.

Figuring out the role of protease in HIV budding

Inside a cell infected by HIV, new virus particles are constructed largely with a protein named Gag. Protease enzymes are incorporated into new viral particles as they are built, and are thought to be activated after the new particles “bud” out of infected cell and then break off from it.

The particles start to bud from the host cell in a saclike container called a vesicle, the neck of which eventually separates from the outer membrane of the infected cell. “Once the particles are released, the proteases are activated and the particles transform into mature HIV, which is infectious,” Saffarian says.

“There is an internal mechanism that dictates activation of the protease, which is not well understood,” he adds. “We found that if we slow the budding process, the protease activates while the HIV particle is still connected to the outer membrane of host [infected] cell. As a result, it chews out all the proteins inside the budding HIV particle, and those essential enzymes and proteins leak back into the host cell. The particle continues to bud out and release from the cell, but it is not infectious anymore because it doesn’t have the enzymes it needs to mature.”

Budding HIV needs ESCRTs

The scientists found they could slow HIV particles from budding out of cells by interfering with how they interact with proteins named ESCRTs (pronounced “escorts”), or “endosomal sorting complexes required for transport.”

ESCRTs are involved in helping pinch off budding HIV particles – essentially cutting them from the infected host cell.

Saffarian says scientific dogma long has held “that messing up the interactions of the virus with ESCRTs results in budding HIV particles permanently getting stuck on the host cell membrane instead of releasing.” Bendjennat says several studies in recent years indicated that the particles do get released, casting some doubt on the long held dogma.

The new study’s significance “is about the molecular mechanism: When the ESCRT machinery is altered, there is production of viruslike particles that are noninfectious,” he says. “This study explains the molecular mechanism of that.”

“We found HIV still releases even when early ESCRT interactions are intentionally compromised, however, with a delay,” Saffarian says. “They are stuck for a while and then they release. And by being stuck for a while, they lose their internal enzymes due to early protease activation and lose their infectivity.”

Bendjennat says by delaying virus budding and speeding “when the protease gets activated, we are now capable of using it to make new released viruses noninfectious”

How the research was done

The experiments used human skin cells grown in tissue culture. It already was known that new HIV particles assemble the same way whether the infected host cell is a skin cell, certain other cells or the T-cell white blood cell infected by the virus to cause AIDS. The experiments involved both live HIV and so-called viruslike particles.

Bendjennat and Saffarian genetically engineered mutant Gag proteins. A single HIV particle is made of some 2,000 Gag proteins and 120 copies of proteins known as Gag-Pol, as well as genetic information in the form of RNA. Pol includes protease, reverse transcriptase and integrase – the proteins HIV uses to replicate.

The mutant Gag proteins were designed to interact abnormally with two different ESCRT proteins, named ALIX and Tsg101.

A new HIV particle normally takes five minutes to release from an infected cell.

When the researchers interfered with ALIX, release was delayed 75 minutes, reducing by half the infectivity of the new virus particle. When the scientists interfered with Tsg101, release was delayed 10 hours and new HIV particles were not infectious.

The scientists also showed that how fast an HIV particle releases from an infected cell depends on how much enzyme cargo it carries in the form of Pol proteins. By interfering with ESCRT proteins during virus-release experiments with viruslike particles made only of Gag protein but none of the normal Pol enzymes, the 75-minute delay shrank to only 20 minutes, and the 10-hour delay shrank to only 50 minutes.

“When the cargo is large, the virus particle needs more help from the ESCRTs to release on a timely fashion,” Saffarian says.

Because HIV carries a large cargo, it depends on ESCRTs to release from an infected cell, so ESCRTs are good targets for drugs to delay release and let HIV proteases leak back into the host cell, making new HIV particles noninfectious, he says.

Bendjennat says other researchers already are looking for drugs to block ESCRT proteins in a way that would prevent the “neck” of the budding HIV particle from pinching off or closing, thus keeping it connected to the infected cell. But he says the same ESCRTs are needed for cell survival, so such drugs would be toxic.

Instead, the new study suggests the right approach is to use low-potency ESCRT-inhibiting drugs that delay HIV release instead of blocking it, rendering it noninfectious with fewer toxic side effects, he adds.

Narcotic painkillers prolong pain in rats, says CU-Boulder study

The dark side of painkillers – their dramatic increase in use and ability to trigger abuse, addiction and thousands of fatal overdoses annually in the United States is in the news virtually every day.

Brace for another shot across the bow: Opioids like morphine have now been shown to paradoxically cause an increase in chronic pain in lab rats, findings that could have far-reaching implications for humans, says a new study led by the University of Colorado Boulder.

Led by CU-Boulder Assistant Research Professor Peter Grace and Distinguished Professor Linda Watkins, the study showed that just a few days of morphine treatment caused chronic pain that went on for several months by exacerbating the release of pain signals from specific immune cells in the spinal cord. The results suggest that the recent escalation of opioid prescriptions in humans may be a contributor to chronic pain, said Grace.

“We are showing for the first time that even a brief exposure to opioids can have long-term negative effects on pain,” said Grace, who is a faculty member along with Watkins in CU-Boulder’s Department of Psychology and Neuroscience. “We found the treatment was contributing to the problem.”

A paper on the study was published May 30 in the Proceedings of the National Academy of Sciences.

The study showed that a peripheral nerve injury in rats sends a message from damaged nerve cells to spinal cord immune cells known as glial cells, which normally act as “housekeepers” to clear out unwanted debris and microorganisms. The first signal of pain sends glial cells into an alert mode, priming them for further action.

“I look at it like turning up a dimmer switch on the spinal cord,” said Grace.

When the injury was treated with just five days of opioids the glial cells went into overdrive, triggering a cascade of actions, including spinal cord inflammation. Watkins said the initial pain signals to the spinal cord and the subsequent morphine-induced treatment is a two-hit process, which she likened to slapping a person’s face.

“You might get away with the first slap, but not the second,” she said. “This one-two hit causes the glial cells to explode into action, making pain neurons go wild.”

The team discovered that the pain signals from a peripheral injury combined with subsequent morphine treatment worked together to cause a glial cell signaling cascade. The cascade produces a cell signal from a protein called interleukin-1beta (IL-1b), which increases the activity of pain-responsive nerve cells in the spinal cord and brain. That can cause increases in pain duration lasting several months.

“The implications for people taking opioids like morphine, oxycodone and methadone are great, since we show the short-term decision to take such opioids can have devastating consequences of making pain worse and longer lasting,” said Watkins. “This is a very ugly side to opioids that had not been recognized before.”

Roughly 20,000 Americans died in 2015 from overdoses of prescription opioid pain relievers, according to the National Institute on Drug Abuse.

On the up side, the researchers have found ways to block specific receptors on glial cells that recognize opioids. This could allow for some pain relief while potentially preventing chronic pain. The team used a designer drug technology known as DREADD to selectively turn off targeted glial cells, something that has not been done before, said Grace.

Hunting for the brain’s opioid addiction switch

New research by Steven Laviolette’s research team at Western University is contributing to a better understanding of the ways opiate-class drugs modify brain circuits to drive the addiction cycle. Using rodent models of opiate addiction, Dr. Laviolette’s research has shown that opiates affect pathways of associative memory formation in multiple ways, both at the level of anatomy (connections between neurons) and at the molecular levels (how molecules inside the brain affect these connections). The identification of these opiate-induced changes offers the best hope for developing more effective pharmacological targets and therapies to prevent or reverse the effect of opiate exposure and addiction. These results were presented at the 10th Annual Canadian Neuroscience Meeting, that took place  in Toronto, Canada.

“Developing more effective opiate addiction treatments will require a change in the way we view the effects of opiates on the brain. Instead of addiction being a chronic, permanent disease, recent evidence is showing that addiction is controlled by molecular switching mechanisms in the brain, that can be turned on or off with the right interventions” says Dr. Steven Laviolette.

Addiction to opiates is spreading and increasing exponentially, and is currently estimated to affect 15.5 million people worldwide. Opiate drugs’ addictive properties are largely due to the ability of this class of drugs to produce powerful memories associated with the intense experience of pleasure and euphoria they cause. Environmental reminders triggering the recall of these memories can cause a relapse, and these memories can be considered the primary driver of the addiction cycle, from chronic use, to withdrawal and then memory-triggered relapse. For decades, clinical and pre-clinical research considered that opiate consumption caused permanent changes in the brain’s reward circuits, resulting in a persistent vulnerability to relapse. However, more recent investigations have shown that opiates induce changes in multiple brain circuits, including reward and memory circuits, and that these changes are not static, but rather that many drug-induced adaptations could be reversed.

“A critical challenge for addiction research is identifying the precise molecular brain changes caused by addictive drugs like heroin or prescription narcotics”, says Dr. Laviolette. “Once we understand this process, we can develop more effective pharmacological interventions to prevent or reverse them”

Among the targets identified by Dr. Laviolette are receptors and other proteins involved in signalling of a neurotransmitter called dopamine. More specifically, his work has shown that dopamine signalling in two connected brain regions involved in opiate-related memory processing, called the Basolateral Amygdala (BLA), a region deep within the brain, and the medial prefrontal cortex (mPFC), located near the surface of the brain, is switched by opiate exposure. His research shows that in animals that are opiate naïve, never previously exposed to opiates, the reward memory associated with opiates requires a dopamine receptor called D1R in the BLA, and a signalling molecule called extracellular signal-related kinase 1/2 (ERK1/2). Following chronic opiate exposure, however, opiate reward memory formation becomes independent of D1R, and rather depends on a second dopamine receptor, called D2r, and a protein called CaMKII. As CaMKII expression has been associated with consolidation and permanence of memories in other brain regions, this switch may reflect the formation of a stronger and more stable opiate reward memory.

Interestingly, when Dr. Laviolette’s team looked at dopamine signaling inside another brain region also involved in opiate related memory procession, and located closer to the surface of the brain, the mPFC, they found that this signaling was also switched by opiate exposure, but opposite to what was observed in the BLA. In the mPFC, opiate naïve signaling requires CaMKII, while it did not in opiate habituated animals.

Taken together, these results highlight the precise changes and adaptations that occur in the brain following opiate exposure and development of addiction. New pharmacological approaches to target these changes will provide much needed and more effective treatments to reduce the power of drug-related associative memories that drive opiate addiction.