MULTIPLE TYPES OF RESISTANCE TO NEW LUNG CANCER DRUG IDENTIFIED

After identifying three different types of resistance to a promising investigational lung cancer drug in a phase 1 trial, a team of researchers led by Dana-Farber Cancer Institute scientists say new targeted inhibitors and combinations are urgently needed to stay ahead of tumors’ constant and varied molecular shape-shifting.

The researchers, including scientists from pharmaceutical company AstraZeneca, report in an article that was published as an advanced online publication in Nature Medicine on May 4, that their findings indicate “an underappreciated genomic heterogeneity” in mechanisms of resistance to tyrosine kinase inhibitor (TKI) drugs that target the Epidermal Growth Factor Receptor (EGFR) mutation that drive some cases of non-small cell lung cancer (NSCLC).

“If resistance that is this complex is constantly evolving before us, it may mean we need multiple targeted therapies in combination to stay ahead of the resistant cancer,” said Geoffrey Oxnard, MD, a thoracic oncologist and lung cancer researcher at Dana-Farber and senior author of the report.

Since the initial discovery of EGFR mutations in lung cancer 10 years ago, EGFR targeted therapies such as erlotinib (Tarceva) and afatinib (Gilotrif) have become a fundamental component of lung cancer therapy. Though they can induce dramatic responses, they tend to lose their effectiveness after nine to 14 months of treatment because of the development of resistance. The most common cause of drug resistance is the development of a second EGFR mutation known as T790-M.

To fight back, pharmaceutical companies are developing and testing next-generation inhibitors aimed at overcoming the T790-M resistance mutation. One such drug, the AstraZeneca compound AZD9291, is showing promise against resistant NSCLC in the ongoing phase 1 AURA clinical trial. In the April 30 issue of the New England Journal of Medicine, Dana-Farber’s Pasi Jänne, MD, PhD, and colleagues reported that AZD9291 shrank lung tumors in 61 percent of patients whose cancers had developed the T790-M resistance mutation. The median progression-free survival in these patients was 10 months.

In the new Nature Medicine study, Oxnard and colleagues looked for mechanisms of resistance to AZD9291 by analyzing liquid biopsies from some of the first patients in the clinical trial whose disease progressed despite treatment with the drug. In effect, they were spying on the cancer’s next strategy for evading the new drug.

Rather than wait for tumor biopsy samples to become available, scientists captured “cell-free” DNA shed into the bloodstream by the tumor cells. Oxnard, working with researchers from the Belfer Institute for Applied Cancer Science at Dana-Farber, had recently developed an assay to detect and quantify EGFR mutations in cell-free DNA. By testing blood specimens at different times during treatment, the scientists identified that three subtypes of resistance emerged.

In some patients, the cancer cells carrying the T790-M mutation acquired an additional EGFR mutation not seen before, labeled C797S, which blocked the AZD9291 from docking to the tumor cells, and causing the disease to advance. In some other patients, the drug failed to eliminate cells with the T790-M resistance mutation – but the C797S mutation was not the culprit. In still other patients, the cancer cells progressed, but the AZD9291 appeared to have eliminated the T790-M resistance mutation, suggesting some other resistance mechanism had taken control. The findings “highlight the need for novel strategies to inhibit EGFR even in the presence of this [C797S] mutation,” the authors said. That might mean designing new drugs that block the effect of the mutation.

While EGFR inhibitors have lengthened lives and improved outcomes in patients with advanced NSCLC, experience has shown that resistance inevitably develops. “The quicker we can learn about drug resistance, the faster we can overcome it,” said Oxnard. He said the new study demonstrates the power of liquid biopsies of resistant cancers to identify the biological causes and – in the present case – report those mechanisms simultaneously with the publication of the clinical trial results for a new drug.

First author of the report is Kenneth S. Thress, PhD, of AstraZeneca. Other authors include Jänne and Cloud Paweletz, PhD, both of the Department of Medical Oncology and the Belfer Institute at Dana-Farber. Study researchers include Yanan Kuang, PhD, also of the Department of Medical Oncology and the Belfer Institute and Dalia Ercan and Sarah Matthews at Dana-Farber.

The study was supported by the National Cancer Institute grants R01CA135257, R01CA114465, and P01CA154303; and AstraZeneca.

Dana-Farber Cancer Institute

DRUG DISCOVERY FOR PARKINSON’S DISEASE: LCSB RESEARCHERS GROW NEURONS IN 3-D

The progressive loss of neurons in the brain of Parkinson’s patients is slow yet inexorable. So far, there are no drugs that can halt this insidious process. Researchers at the Luxembourg Centre for Systems Biomedicine (LCSB) of the University of Luxembourg have now managed to grow the types of neurons affected starting from neuronal stem cells in a three-dimensional cell culture system. The scientists working with Dr. Ronan Fleming of the LCSB research group Systems Biochemistry are confident this system could greatly facilitate the continuing search for therapeutic agents in future as it models the natural conditions in the brain more realistically than other systems available so far. It is also significantly cheaper to employ in the laboratory. The results were recently published in the journal “Lab on a Chip” (doi: 10.1039/C5LC00180C).

Parkinson’s disease is characterised in particular by the death of dopamine-producing neurons in the Substantia nigra of the midbrain. It is already possible to grow these dopaminergic neurons in cell cultures. “But most such cell cultures are two-dimensional, with the cells growing along the base of a petri dish, for example,” group leader Fleming explains. “Instead, we have the neurons grow in a gel that yields a far better model of their natural, three-dimensional environment.”

As the starting point for cultivating the target neurons, the scientists use ordinary skin cells. They convert these through conventional methods into induced pluripotent stem cells, or iPSCs for short. For the development of this technology Japanese scientist Shinya Yamanaka was awarded the Nobel Prize for Physiology or Medicine in 2012 together with John Gurdon. “By adding suitable growth factors, the iPSCs can then be converted in a second step into neural stem cells,” says Prof. Jens Schwamborn, head of the LCSB research group Developmental & Cellular Biology, which is responsible for the differentiation of the cells. “These are the starting cells we use in the microfluidic culture.”

The researchers first mix the cells with a liquid, which they then fill into little test vessels called bioreactors. “You can imagine such a bioreactor as a tunnel separated down the middle by a flat barrier,” LCSB researcher Edinson Lucumi Moreno, first author of the study, explains. “One side of the tunnel we load the liquid with the cells, where it hardens into a gel under controlled temperatures. The other side we load with a medium to which we can add nutrients and substances for further differentiation of the neuronal stem cells as required.”

After only a few hours, the researchers already observe changes in the neuronal stem cells: The cells begin to form little protrusions, which develop over the following days into axons and dendrites - the long extensions typical of neurons. After 30 days, 91 percent of the cells are neurons, about 20 percent of which are the desired dopaminergic neurons. This has been confirmed in morphological and immunological tests.

One of the major advantages of this 3D cell culture system is that it can already be automated in its present form: The bioreactors are placed on commercially available plates that can be processed and read out by laboratory robots. “In drug development, dozens of chemical substances can therefore be tested for possible therapeutic effects in a single step,” says Ronan Fleming. “Because we use far smaller amounts of substances than in conventional cell culture systems, the costs drop to about one tenth the usual.”

A further advantage is that the bioreactors can be loaded with cells originating from the skin cells of individual Parkinson’s patients. “This is an important step towards personalised drug development,” Fleming asserts. As a next step, Fleming’s team and their international collaborators want to study cells from patients and to test potential active pharmaceutical ingredients. Promising substances will then be tested in mice.

University of Luxembourg

DNDI AND FOUR PHARMACEUTICAL COMPANIES ANNOUNCE ‘DRUG DISCOVERY BOOSTER’ EXPERIMENT TO ACCELERATE AND EXPAND DISCOVERY OF NEW DRUGS FOR TWO HIGHLY NEGLECTED DISEASES – LEISHMANIASIS AND CHAGAS DISEASE

The Drugs for Neglected Diseases initiative (DNDi) and four pharmaceutical firms, Eisai Co Ltd, Shionogi & Co Ltd, Takeda Pharmaceutical Ltd, and AstraZeneca plc have announced the start of a ground-breaking initiative to accelerate and cut the cost of early stage drug discovery for two of the world’s most neglected diseases, leishmaniasis and Chagas disease. The ‘Neglected Tropical Diseases Drug Discovery Booster’ consortium, through a carefully engineered modus operandi, will circumvent early stage commercial barriers between the four pharmaceutical participants, allowing DNDi, for the first time, to search millions of unique compounds simultaneously, in the hunt for new treatment leads for leishmaniasis and Chagas disease.

‘This experimental approach to radically modernize drug development for neglected diseases is the result of a decade of growing partnerships with pharmaceutical companies’, said Dr Bernard Pécoul, Executive Director of DNDi. ‘This experiment could significantly reduce the time and money it takes to find new, promising treatment leads, and echoes the great potential of innovative research and development collaborations.’

Traditionally, early stage drug discovery to find new treatments has been an expensive and time-consuming process. To identify promising new compounds for neglected diseases, DNDi has worked bilaterally with a range of pharmaceutical partners, searching through segments of their vast compound libraries, and testing them against infected cells. The Drug Discovery Booster is a new approach, using a multilateral, simultaneous search process across participating companies, thus allowing DNDi access to compounds generated over many decades of research. State-of-the-art technology will be used to pinpoint compounds that have promising characteristics for further testing.

The innovation of the Drug Discovery Booster not only lies in the multilateral approach, but also in the iterative nature of the search, meaning companies will continually examine their libraries for better matches as the search is refined. The Drug Discovery Booster has the potential to cut up to two years from the early drug discovery process, which takes approximately five years or more, and improve cost-efficiency across research activities.

The new process starts with DNDi providing all four companies with a common chemical starting point, the ‘seed’ compound. This compound will have shown promising results against Leishmania or Trypanosoma cruzi, the parasites that cause leishmaniasis and Chagas disease, but may not yet be optimal for use as a future treatment. The four companies will then search their own full collections of high-quality chemical compounds for similar and potentially better molecules, and will select and send the most promising to DNDi, which will then have them screened for potential effectiveness against these two deadly parasitic diseases. The aim is to continually improve the effectiveness of the compounds.

DNDi will then select the best ‘hits’ for further testing. This process will be repeated up to three times, with each new iteration – or round – starting from an improved seed compound identified from within one of the four partner’s collections and shared with all. The initial project goal is to explore the consortium’s compound libraries for at least four promising seed compounds for each disease. It is expected that at least two of the resulting novel series of compounds will move to the next stage of development towards a new medicine.

Any progress or successful new treatment for leishmaniasis or Chagas disease resulting from the Drug Discovery Booster will be attributed to the collective effort of all partners, which have also agreed that no intellectual property barriers will be imposed to a new treatment if successful.

The GHIT Fund, launched in 2013, stimulates and supports such international global health research and development partnerships. Through a grant of EUR 640,000 or 79.5 million Japanese Yen provided to DNDi, GHIT will support the involvement of the three Japanese companies in this project. The remainder of the project, including the involvement of AstraZeneca plc, is being supported by DNDi core funds.

‘The Drug Discovery Booster could be a game-changing milestone in the fight against diseases that destroy the health and livelihoods of the world’s poorest’, said Dr BT Slingsby, GHIT Fund CEO. ‘Industry’s innovative leadership for the most neglected of the neglected diseases will be essential to accelerate progress toward creating life-saving medicines, and GHIT is proud to support the important contribution of Japanese pharmaceutical companies to make this possible.’

Drugs for Neglected Diseases initiative (DNDi)

DISCOVERY PAVES WAY FOR HOMEBREWED DRUGS, PROMPTS CALL FOR REGULATION

Fans of homebrewed beer and backyard distilleries already know how to employ yeast to convert sugar into alcohol. But a research team led by UC Berkeley bioengineers has gone much further by completing key steps needed to turn sugar-fed yeast into a microbial factory for producing morphine and potentially other drugs, including antibiotics and anti-cancer therapeutics.

Over the past decade, a handful of synthetic-biology labs have been working on replicating in microbes a complex, 15-step chemical pathway in the poppy plant to enable production of therapeutic drugs. Research teams have independently recreated different sections of the poppy’s drug pathway using E. coli or yeast, but what had been missing until now were the final steps that would allow a single organism to perform the task from start to finish.

In a new study appearing in the advanced online publication of the journal Nature Chemical Biology, UC Berkeley bioengineer John Dueber teamed up with microbiologist Vincent Martin at Concordia University in Montreal, to overcome that hurdle by replicating the early steps in the pathway in an engineered strain of yeast. They were able to synthesize reticuline, a compound in poppy, from tyrosine, a derivative of glucose.

“What you really want to do from a fermentation perspective is to be able to feed the yeast glucose, which is a cheap sugar source, and have the yeast do all the chemical steps required downstream to make your target therapeutic drug,” said Dueber, the study’s principal investigator and an assistant professor of bioengineering. “With our study, all the steps have been described, and it’s now a matter of linking them together and scaling up the process. It’s not a trivial challenge, but it’s doable.”

Paving the path from plants to microbes

The qualities that make the poppy plant pathway so challenging are the same ones that make it such an attractive target for research. It is complex, but it is the foundation upon which researchers can build new therapeutics. Benzylisoquinoline alkaloids, or BIAs, are the class of highly bioactive compounds found in the poppy, and that family includes some 2,500 molecules isolated from plants.

Perhaps the best-known trail in the BIA pathway is the one that leads to the opiates, such as codeine, morphine and thebaine, a precursor to oxycodone and hydrocodone. All are controlled substances. But different trails will lead to the antispasmodic papaverine or to the antibiotic precursor dihydrosanguinarine.

“Plants have slow growth cycles, so it’s hard to fully explore all the possible chemicals that can be made from the BIA pathway by genetically engineering the poppy,” said study lead author William DeLoache, a UC Berkeley Ph.D. student in bioengineering. “Moving the BIA pathway to microbes dramatically reduces the cost of drug discovery. We can easily manipulate and tune the DNA of the yeast and quickly test the results.”

The researchers found that by repurposing an enzyme from beets that is naturally used in the production of their vibrant pigments, they could coax yeast to convert tyrosine, an amino acid readily derived from glucose, into dopamine.

With help from the lab of Concordia University’s Vincent Martin, the researchers were able to reconstitute the full seven-enzyme pathway from tyrosine to reticuline in yeast.

“Getting to reticuline is critical because from there, the molecular steps that produce codeine and morphine from reticuline have already been described in yeast,” said Martin, a professor of microbial genomics and engineering. “Also, reticuline is a molecular hub in the BIA pathway. From there, we can explore many different paths to other potential drugs, not just opiates.”

Red flag for regulators

The study authors noted that the discovery dramatically speeds up the clock for when homebrewing drugs could become a reality, and they are calling for regulators and law enforcement officials to pay attention.

“We’re likely looking at a timeline of a couple of years, not a decade or more, when sugar-fed yeast could reliably produce a controlled substance,” said Dueber. “The time is now to think about policies to address this area of research. The field is moving surprisingly fast, and we need to be out in front so that we can mitigate the potential for abuse.”

In a commentary to be published in Nature and timed with the publication of this study, policy analysts call for urgent regulation of this new technology. They highlight the many benefits of this work, but they also point out that “individuals with access to the yeast strain and basic skills in fermentation would be able to grow the yeast using the equivalent of a homebrew kit.”

They recommend restricting engineered yeast strains to licensed facilities and to authorized researchers, noting that it would be difficult to detect and control the illicit transport of such strains.

While such controls may help, Dueber said, “An additional concern is that once the knowledge of how to create an opiate-producing strain is out there, anyone trained in basic molecular biology could theoretically build it.”

Another target for regulation would be the companies that synthesize and sell DNA sequences. “Restrictions are already in place for sequences tied to pathogenic organisms, like smallpox,” said DeLoache. “But maybe it’s time we also look at sequences for producing controlled substances.”

Other co-authors on this study are Zachary Russ and Andrew Gonzales of UC Berkeley’s Department of Bioengineering, and Lauren Narcross of Concordia University’s Department of Biology.

Berkeley University of California

SCRIPPS FLORIDA SCIENTISTS IDENTIFY A POTENTIAL NEW TREATMENT FOR OSTEOPOROSIS

Scientists from the Florida campus of The Scripps Research Institute (TSRI) have identified a new therapeutic approach that, while still preliminary, could promote the development of new bone-forming cells in patients suffering from bone loss.

The study, published in the journal Nature Communications, focused on a protein called PPARy (known as the master regulator of fat) and its impact on the fate of stem cells derived from bone marrow (“mesenchymal stem cells”). Since these mesenchymal stem cells can develop into several different cell types—including fat, connective tissues, bone and cartilage—they have a number of potentially important therapeutic applications.

The scientists knew that a partial loss of PPARy in a genetically modified mouse model led to increased bone formation. To see if they could mimic that effect using a drug candidate, the researchers combined a variety of structural biology approaches to rationally design a new compound that could repress the biological activity of PPARy.

The results showed that when human mesenchymal stem cells were treated with the new compound, which they called SR2595 (SR=Scripps Research), there was a statistically significant increase in osteoblast formation, a cell type known to form bone.

“These findings demonstrate for the first time a new therapeutic application for drugs targeting PPARy, which has been the focus of efforts to develop insulin sensitizers to treat type 2 diabetes,” said Patrick Griffin, chair of the Department of Molecular Therapeutics and director of the Translational Research Institute at Scripps Florida. “We have already demonstrated SR2595 has suitable properties for testing in mice; the next step is to perform an in-depth analysis of the drug’s efficacy in animal models of bone loss, aging, obesity and diabetes.”

In addition to identifying a potential new therapeutic for bone loss, the study may have even broader implications.

“Because PPARG is so closely related to several proteins with known roles in disease, we can potentially apply these structural insights to design new compounds for a variety of therapeutic applications,” said David P. Marciano, first author of the study, a recent graduate of TSRI’s PhD program and former member of the Griffin lab. “In addition, we now better understand how natural molecules in our bodies regulate metabolic and bone homeostasis, and how unwanted changes can underlie the pathogenesis of a disease.” Marciano will focus on this subject in his postdoctoral work in the Department of Genetics at Stanford University.

The work was supported by the National Institutes of Health (grants DK08026, MH084512, OD018254-01, DK097890, DK103116 and DK101871).

Scripps Research Institute

MIGRAINE COMMUNITY SEES PROMISE IN NEW CLASS OF DRUGS; OPTIMISTIC FOR RELIEF FOR MILLIONS OF SUFFERERS

Migraine researchers and clinicians are growing excited about a new class of drugs called Calcitonin Gene-Related Peptide (CGRP) monoclonal antibodies, which are showing promise in treating high-frequency episodic migraine and chronic migraine.

“This development is a transformative moment in migraine treatment,” said Peter J. Goadsby, MD, PhD, who is chair of the scientific program of the American Headache Society’s annual Scientific Meeting. Dr. Goadsby is Chief of the UCSF Headache Center, and one of the world’s leading headache treatment experts and researchers.

“There’s no question that we need something better,” he said. “In fact, for prevention we really need something designed specifically for migraine,” he said, noting that there has not been a new class of anti-migraine drugs since the development and marketing of triptans in 1991 and they are not preventives, just designed to treat migraine attacks.

“Up till now, migraine patients have had limited choices for preventive treatment. Now four pharmaceutical companies are showing positive results in human trials targeting CGRP mechanisms,” he said.

The new class of therapeutic agents appears to reduce elevated levels of the peptide known as calcitonin gene-related peptide (CGRP), a key driver of migraine pain.

Versions of anti-CGRP therapies are being tested by Alder Pharmaceuticals, Amgen, Eli Lilly and Company, and Teva Pharmaceuticals.

In Phase IIb trials (studies conducted patients with migraine) data presented at the American Headache Society meeting by Teva reported for the first time that its drug, as a preventive treatment of high frequency episodic migraine, achieved a significant reducti on in the number of headache hours after one week, with more than half of patients in each arm experiencing a 50% or greater reduction in headache frequency. Lilly presented, for the first time, Phase II data in episodic migraine that establishes the efficacy of their medicine against placebo with monthly administration across a range of doses. Amgen presented Phase II data for its anti-CGRP product that showed that the drug reduced the number of migraine days by 50% in about half the treated patients after 12 weeks. Alder Pharmaceutical, the fourth player in the CGRP race, is also developing an anti-CGRP drug with positive phase II data published, and did not present further data at the meeting.

“The potential of these new compounds is enormous and gives us real hope that effective specific treatments for migraine may be on the near horizon,” Dr. Goadsby said. “The development of CGRP antibodies offers the simple, yet elegant and long awaited option for migraine patients to finally be treated with migraine preventives; it’s a truly landmark development.”

American Headache Society