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June 22-24: Free Registration for AACR Members, the Cancer Community, and the Public
This virtual meeting will feature more than 120 sessions and 4,000 e-posters, including sessions on cancer health disparities and the impact of COVID-19 on clinical trials
This Virtual Meeting is Part II of the AACR Annual Meeting. Part I was held online in April and was centered only on clinical findings. This Part II of the virtual meeting will contain all the Sessions and Abstracts pertaining to basic and translational cancer research as well as clinical trial findings.
The prestigious Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Research was established in 1997 to annually recognize a scientist of international renown who has made a major scientific discovery in basic cancer research OR who has made significant contributions to translational cancer research; who continues to be active in cancer research and has a record of recent, noteworthy publications; and whose ongoing work holds promise for continued substantive contributions to progress in the field of cancer. For more information regarding the 2020 award recipient go to aacr.org/awards.
Princess Anne Margaret Cancer Center, Toronto, Ontario
For determining how stem cells contribute to normal and leukemic hematopoeisis
not every cancer cell equal in their Cancer Hallmarks
how do we monitor and measure clonal dynamics
Barnie Clarkson did pivotal work on this
most cancer cells are post mitotic but minor populations of cells were dormant and survive chemotherapy
only one cell is 1 in a million can regenerate and transplantable in mice and experiments with flow cytometry resolved the question of potency and repopulation of only small percentage of cells and undergo long term clonal population
so instead of going to cell lines and using thousands of shRNA looked at clinical data and deconvoluted the genetic information (RNASeq data) to determine progenitor and mature populations (how much is stem and how much is mature populations)
in leukemic patients they have seen massive expansion of a single stem cell population so only need one cell in AML if the stem cells have the mutational hits early on in their development
finding the “seeds of relapse”: finding the small subpopulation of stem cells that will relapse
they looked in BALL;; there are cells resistant to l-aspariginase, dexamethasone, and vincristine
a lot of OXPHOS related genes (in DRIs) that may be the genes involved in this resistance
it a wonderful note of acknowledgement he dedicated this award to all of his past and present trainees who were the ones, as he said, made this field into what it is and for taking it into directions none of them could forsee
Monday, June 22
1:30 PM – 3:30 PM EDT
Virtual Educational Session
Experimental and Molecular Therapeutics, Drug Development, Cancer Chemistry
How can one continue to deliver innovative medicines to patients when biological targets are becoming ever scarcer and less amenable to therapeutic intervention? Are there sound strategies in place that can clear the path to targets previously considered “undruggable”? Recent advances in lead finding methods and novel technologies such as covalent screening and targeted protein degradation have enriched the toolbox at the disposal of drug discovery scientists to expand the druggable ta
Stefan N Gradl, Elena S Koltun, Scott D Edmondson, Matthew A. Marx, Joachim Rudolph
Cancer researchers are faced with a deluge of high-throughput data. Using these data to advance understanding of cancer biology and improve clinical outcomes increasingly requires effective use of computational and informatics tools. This session will introduce informatics resources that support the data management, analysis, visualization, and interpretation. The primary focus will be on high-throughput genomic data and imaging data. Participants will be introduced to fundamental concepts
Rachel Karchin, Daniel Marcus, Andriy Fedorov, Obi Lee Griffith
Precision medicine refers to the use of prevention and treatment strategies that are tailored to the unique features of each individual and their disease. In the context of cancer this might involve the identification of specific mutations shown to predict response to a targeted therapy. The biomedical literature describing these associations is large and growing rapidly. Currently these interpretations exist largely in private or encumbered databases resulting in extensive repetition of effort.
CIViC’s Role in Precision Medicine
Realizing precision medicine will require this information to be centralized, debated and interpreted for application in the clinic. CIViC is an open access, open source, community-driven web resource for Clinical Interpretation of Variants in Cancer. Our goal is to enable precision medicine by providing an educational forum for dissemination of knowledge and active discussion of the clinical significance of cancer genome alterations. For more details refer to the 2017 CIViC publication in Nature Genetics.
U24 funding announced: We are excited to announce that the Informatics Technology for Cancer Research (ICTR) program of the National Cancer Institute (NCI) has awarded funding to the CIViC team! Starting this year, a five-year, $3.7 million U24 award (CA237719), will support CIViC to develop Standardized and Genome-Wide Clinical Interpretation of Complex Genotypes for Cancer Precision Medicine.
Informatics tools for high-throughput analysis of cancer mutations
Rachel Karchin
CRAVAT is a platform to determine, categorize, and curate cancer mutations and cancer related variants
adding new tools used to be hard but having an open architecture allows for modular growth and easy integration of other tools
so they are actively making an open network using social media
While LOD has had some uptake across the web, the number of databases using this protocol compared to the other technologies is still modest. But whether or not we use LOD, we do need to ensure that databases are designed specifically for the web and for reuse by humans and machines. To provide guidance for creating such databases independent of the technology used, the FAIR principles were issued through FORCE11: the Future of Research Communications and e-Scholarship. The FAIR principles put forth characteristics that contemporary data resources, tools, vocabularies and infrastructures should exhibit to assist discovery and reuse by third-parties through the web. Wilkinson et al.,2016. FAIR stands for: Findable, Accessible, Interoperable and Re-usable. The definition of FAIR is provided in Table 1:
Number
Principle
F
Findable
F1
(meta)data are assigned a globally unique and persistent identifier
F2
data are described with rich metadata
F3
metadata clearly and explicitly include the identifier of the data it describes
F4
(meta)data are registered or indexed in a searchable resource
A
Accessible
A1
(meta)data are retrievable by their identifier using a standardized communications protocol
A1.1
the protocol is open, free, and universally implementable
A1.2
the protocol allows for an authentication and authorization procedure, where necessary
A2
metadata are accessible, even when the data are no longer available
I
Interoperable
I1
(meta)data use a formal, accessible, shared, and broadly applicable language for knowledge representation.
I2
(meta)data use vocabularies that follow FAIR principles
I3
(meta)data include qualified references to other (meta)data
R
Reusable
R1
meta(data) are richly described with a plurality of accurate and relevant attributes
R1.1
(meta)data are released with a clear and accessible data usage license
R1.2
(meta)data are associated with detailed provenance
R1.3
(meta)data meet domain-relevant community standards
A detailed explanation of each of these is included in the Wilkinson et al., 2016 article, and the Dutch Techcenter for Life Sciences has a set of excellent tutorials, so we won’t go into too much detail here.
for outside vendors to access their data, vendors would need a signed Material Transfer Agreement but NCI had formulated a framework to facilitate sharing of data using a DIACOM standard for imaging data
Monday, June 22
1:30 PM – 3:01 PM EDT
Virtual Educational Session
Experimental and Molecular Therapeutics, Cancer Chemistry, Drug Development, Immunology
The engineering and physical science disciplines have been increasingly involved in the development of new approaches to investigate, diagnose, and treat cancer. This session will address many of these efforts, including therapeutic methods such as improvements in drug delivery/targeting, new drugs and devices to effect immunomodulation and to synergize with immunotherapies, and intraoperative probes to improve surgical interventions. Imaging technologies and probes, sensors, and bioma
Claudia Fischbach, Ronit Satchi-Fainaro, Daniel A Heller
How should we think about exceptional and super responders to cancer therapy? What biologic insights might ensue from considering these cases? What are ways in which considering super responders may lead to misleading conclusions? What are the pros and cons of the quest to locate exceptional and super responders?
Alice P Chen, Vinay K Prasad, Celeste Leigh Pearce
The reprogramming of cellular metabolism is a hallmark feature observed across cancers. Contemporary research in this area has led to the discovery of tumor-specific metabolic mechanisms and illustrated ways that these can serve as selective, exploitable vulnerabilities. In this session, four international experts in tumor metabolism will discuss new findings concerning the rewiring of metabolic programs in cancer that support metabolic fitness, biosynthesis, redox balance, and the reg
Costas Andreas Lyssiotis, Gina M DeNicola, Ayelet Erez, Oliver Maddocks
The retina is responsible for capturing images from the visual field. Retinitis pigmentosa, which refers to a group of inherited diseases that cause retinal degeneration, causes a gradual decline in vision because retinal photoreceptor cells (rods and cones) die. Images on the left are courtesy of the National Eye Institute, NIH; image on the right is courtesy of Robert Fariss, Ph.D., and Ann Milam, Ph.D., National Eye Institute, NIH.
Metabolomics, the comprehensive evaluation of the products of cellular processes, can provide new findings and insight in a vast array of diseases and dysfunctions. Though promising, metabolomics lacks the standing of genomics or proteomics. It is, in a manner of speaking, the new kid on the “omics” block.
Even though metabolomics is still an emerging discipline, at least some quarters are giving it a warm welcome. For example, metabolomics is being advanced by the Common Fund, an initiate of the National Institutes of Health (NIH). The Common Fund has established six national metabolomics cores. In addition, individual agencies within NIH, such as the National Institute of Environmental Health Sciences (NIEHS), are releasing solicitations focused on growing more detailed metabolomics programs.
Whether metabolomic studies are undertaken with or without public support, they share certain characteristics and challenges. Untargeted or broad-spectrum studies are used for hypotheses generation, whereas targeted studies probe specific compounds or pathways. Reproducibility is a major challenge in the field; many studies cannot be reproduced in larger cohorts. Carefully defined guidance and standard operating procedures for sample collection and processing are needed.
While these challenges are being addressed, researchers are patiently amassing metabolomic insights in several areas, such as retinal diseases, neurodegenerative diseases, and autoimmune diseases. In addition, metabolomic sleuths are availing themselves of a growing selection of investigative tools.
A Metabolomic Eye on Retinal Degeneration
The retina has one of the highest metabolic activities of any tissue in the body and is composed of multiple cell types. This fact suggests that metabolomics might be helpful in understanding retinal degeneration. At least, that’s what occurred to Ellen Weiss, Ph.D., a professor of cell biology and physiology at the University of North Carolina School of Medicine at Chapel Hill. To explore this possibility, Dr. Weiss began collaborating with Susan Sumner, Ph.D., director of systems and translational sciences at RTI International.
Retinal degeneration is often studied through the use of genetic-mouse models that mimic the disease in humans. In the model used by Dr. Weiss, cells with a disease-causing mutation are the major light-sensing cells that degenerate during the disease. Individuals with the same or a similar genetic mutation will initially lose dim-light vision then, ultimately, bright-light vision and color vision.
Wild-type and mutant phenotypes, as well as dark- and light-raised animals, were compared, since retinal degeneration is exacerbated by light in this genetic model. Retinas were collected as early as day 18, prior to symptomatic disease, and analyzed. Although data analysis is ongoing, distinct differences have emerged between the phenotypes as well as between dark- and light-raised animals.
“There is a clear increase in oxidative stress in both light-raised groups but to a larger extent in the mutant phenotype,” reports Dr. Weiss. “There are global changes in metabolites that suggest mitochondrial dysfunction, and dramatic changes in lipid profiles. Now we need to understand how these metabolites are involved in this eye disease and the relevance of these perturbations.”
For example, the glial cells in the retina that upregulate a number of proteins in response to stress to attempt to save the retina are as likely as the light-receptive neurons to undergo metabolic changes.
“One of the challenges in metabolomics studies is assigning the signals that represent the metabolites or compounds in the samples,” notes Dr. Sumner. “Signals may be ‘unknown unknowns,’ compounds that have never been identified before, or ‘known unknowns,’ compounds that are known but that have not yet been assigned in the biological matrix.”
Internal and external libraries, such as the Human Metabolome Dictionary, are used to match signals. Whether or not a match exists, fragmentation patterns are used to characterize the metabolite, and when possible a standard is obtained to confirm identity. To assist with this process, the NIH Common Fund supports Metabolite Standard Synthesis Cores (MSSCs). RTI International holds an MSSC contract in addition to being a NIH-designated metabolomics core.
Mitochondrial Dysfunction in Alzheimer’s Disease
Alzheimer’s disease (AD) is difficult to diagnose early due to its asymptomatic phase; accurate diagnosis occurs only in postmortem brain tissue. To evaluate familial AD, a rare inherited form of the disease, the laboratory of Eugenia Trushina, Ph.D., associate professor of neurology and associate professor of pharmacology at the Mayo Clinic, uses mouse models to study the disease’s early molecular mechanisms.
Synaptic loss underlies cognitive dysfunction. The length of neurons dictates that mitochondria move within the cell to provide energy at the site of the synapses. An initial finding was that very early on mitochondrial trafficking was affected reducing energy supply to synapses and distant parts of the cell.
During energy production, the major mitochondrial metabolite is ATP, but the organelle also produces many other metabolites, molecules that are implicated in many pathways. One can assume that changes in energy utilization, production, and delivery are associated with some disturbance.
“Our goal,” explains Dr. Trushina, “was to get a proof of concept that we could detect in the blood of AD patients early changes of mitochondria dysfunction or other changes that could be informative of the disease over time.”
A Mayo Clinic aging study involves a cohort of patients, from healthy to those with mild cognitive impairment (MCI) through AD. Patients undergo an annual battery of tests including cognitive function along with blood and cerebrospinal fluid sampling. Metabolic signatures in plasma and cerebrospinal fluid of normal versus various disease stages were compared, and affected mitochondrial and lipid pathways identified in MCI patients that progressed to AD.
“Last year we published on a new compound that goes through the blood/brain barrier, gets into mitochondria, and very specifically, partially inhibits mitochondrial complex I activity, making the cell resistant to oxidative damage,” details Dr. Trushina. “The compound was able to either prevent or slow the disease in the animal familial models.
“Treatment not only reduced levels of amyloid plaques and phosphorylated tau, it also restored mitochondrial transport in neurons. Now we have additional compounds undergoing investigation for safety in humans, and target selectivity and engagement.”
“Mitochondria play a huge role in every aspect of our lives,” Dr. Trushina continues. “The discovery seems counterintuitive, but if mitochondria function is at the heart of AD, it may provide insight into the major sporadic form of the disease.”
Distinguishing Types of Asthma
In children, asthma generally manifests as allergy-induced asthma, or allergic asthma. And allergic asthma has commonalities with allergic dermatitis/eczema, food allergies, and allergic rhinitis. In adults, asthma is more heterogeneous, and distinct and varied subpopulations emerge. Some have nonallergic asthma; some have adult-onset asthma; and some have obesity-, occupational-, or exercise-induced asthma.
Adult asthmatics may have markers of TH2 high verus TH2 low asthma (T helper 2 cell cytokines) and they may respond to various triggers—environmental antigens, occupational antigens, irritants such as perfumes and chlorine, and seasonal allergens. Exercise, too, can trigger asthma.
One measure that can phenotype asthmatics is nitric oxide, an exhaled breath biomarker. Nitric oxide is a smooth muscle relaxant, vasodilator, and bronchodilator that can have anti-inflammatory properties. There is a wide range of values in asthmatics, and a number of values are needed to understand the trend in a particular patient. L-arginine is the amino acid that produces nitric oxide when converted to L-citrulline, a nonessential amino acid.
According to Nicholas Kenyon, M.D., a pulmonary and critical care specialist who is co-director of the University of California, Davis Asthma Network (UCAN), some metabolomic studies suggest that there is a state of L-arginine depletion during asthma attacks or in severe asthma suggesting a lack of substrate to produce nitric oxide. Dr. Kenyon is conducting clinical work on L-arginine supplementation in a double-blind cross-over intervention trial of L-arginine versus placebo. The 50-subject study in severe asthmatics should be concluded in early 2017.
Many new biologic therapies are coming to market to treat asthma; it will be challenging to determine which advanced therapy to provide to which patient. Therapeutics mostly target severe asthma populations and are for patients with evidence of higher numbers of eosinophils in the blood and lung, which include anti-IL-5 and (soon) anti-IL-13, among others.
Tools Development
Waters is developing metabolomics applications that use multivariate statistical methods to highlight compounds of interest. Typically these applications combine separation procedures, accomplished by means of liquid chromatography or gas chromatography (LC or GC), with detection methods that rely on mass spectrometry (MS). To support the identification, quantification, and analysis of LC-MS data, the company provides bioinformatics software. For example, Progenesis QI software can interrogate publicly available databases and process information about isotopic patterns, retention times, and collision cross-sections.
Mass spectrometry (MS) is the gold standard in metabolomics and lipidomics. But there is a limit to what accurate mass and resolution can achieve. For example, neither isobaric nor isomeric species are resolvable solely by MS. New orthogonal analytical tools will allow more confident identifications.
To improve metabolomics separations before MS detection, a post-ionization separation tool, like ion mobility, which is currently used to support traditional UPLC-MS and MS imaging metabolomics protocols, becomes useful. The collision-cross section (CCS), which measures the shape of molecules, can be derived, and it can be used as an additional identification coordinate.
Other new chromatographic tools are under development, such as microflow devices and UltraPerformance Convergence Chromatography (UPC2), which uses liquid CO2 as its mobile phase, to enable new ways of separating chiral metabolites. Both UPC2 and microflow technologies have decreased solvent consumption and waste disposal while maintaining UPLC-quality performance in terms of chromatographic resolution, robustness, and reproducibility.
Informatics tools are also improving. In the latest versions of Waters’ Progenesis software, typical metabolomics identification problems are resolved by allowing interrogation of publicly available databases and scoring according to accurate mass, isotopic pattern, retention time, CCS, and either theoretical or experimental fragments.
MS imaging techniques, such as MALDI and DESI, provide spatial information about the metabolite composition in tissues. These approaches can be used to support and confirm traditional analyses without sample extraction, and they allow image generation without the use of antibodies, similar to immunohistochemistry.
“Ion-mobility tools will soon be implemented for routine use, and the use of extended CCS databases will help with metabolite identification,” comments Giuseppe Astarita Ph.D., principal scientist, Waters. “More applications of ambient ionization MS will emerge, and they will allow direct-sampling analyses at atmospheric pressure with little or no sample preparation, generating real-time molecular fingerprints that can be used to discriminate among phenotypes.”
Microflow Technology
Microflow technology offers sensitivity and robustness. For example, at the Proteomics and Metabolomics Facility, Colorado State University, peptide analysis was typically performed using nanoflow chromatography; however, nanoflow chromatography is slow and technically challenging. Moving to microflow offered significant improvements in robustness and ease-of-use and resulted in improved chromatography without sacrificing sensitivity.
Conversely, small molecule applications were typically performed with analytical-scale chromatography. While this flow regime is extremely robust and fast, it can sometimes be limited in sensitivity. Moving to microflow offered significant improvements in sensitivity, 5- to 10-fold depending on the compound, without sacrificing robustness.
But broad-scale microflow adoption is hampered by a lack of available column chemistries and legacy HPLC or UPLC infrastructure that is not conducive to low-flow operation.
“We utilize microflow technology on all of our tandem quadrupole instruments for targeted quantitative assays,” says Jessica Prenni, Ph.D., director, Proteomics and Metabolomics Facility, Colorado State University. “All of our peptide quantitation is exclusively performed with microflow technology, and many of our small molecule assays. Application examples include endocannabinoids, bile acids and plant phytohormone panels.”
Compound annotation and comparability and transparency in data processing and reporting is a challenge in metabolomics research. Multiple groups are actively working on developing new tools and strategies; common best practices need to be adopted.
The continued growth of open-source spectral databases and new tools for spectral prediction from compound databases will dramatically impact the ability for metabolomics to result in novel discoveries. The move to a systems-level understanding through the combination of various omics data also will have a huge influence and be enabled by the continued development of open-source and user-friendly pathway-analysis tools.
Where Trackless Terrain Once Challenged Biomarker Development, Clearer Paths Are Emerging
Fusion detection can be carried out with traditional opposing primer-based library preparation methods, which require target- and fusion-specific primers that define the region to be sequenced. With these methods, primers are needed that flank the target region and the fusion partner, so only known fusions can be detected. An alternative method, ArcherDX’ Anchored Multiplex PCR (AMP), can be used to detect the target of interest, plus any known and unknown fusion partners. This is because AMP uses target-specific unidirectional primers, along with reverse primers, that hybridize to the sequencing adapter that is ligated to each fragment prior to amplification.
In time, the narrow, tortuous paths followed by pioneers become wider and straighter, whether the pioneers are looking to settle new land or bring new biomarkers to the clinic.
In the case of biomarkers, we’re still at the stage where pioneers need to consult guides and outfitters or, in modern parlance, consultants and technology providers. These hardy souls tend to congregate at events like the Biomarker Conference, which was held recently in San Diego.
At this event, biomarker experts discussed ways to avoid unfortunate detours on the trail from discovery and development to clinical application and regulatory approval. Of particular interest were topics such as the identification of accurate biomarkers, the explication of disease mechanisms, the stratification of patient groups, and the development of standard protocols and assay platforms. In each of these areas, presenters reported progress.
Another crucial subject is the integration of techniques such as next-generation sequencing (NGS). This particular technique has been instrumental in advancing clinical cancer genomics and continues to be the most feasible way of simultaneously interrogating multiple genes for driver mutations.
Enriching nucleic acid libraries for target genes of interest prior to NGS greatly enhances the sensitivity of detecting mutations, as the enriched regions are sequenced multiple times. This is particularly useful when analyzing clinical samples, which generate low amounts of poor-quality nucleic acids.
Most target-enrichment strategies require prior knowledge of both ends of the target region to be sequenced. Therefore, only gene fusions with known partners can be amplified for downstream NGS assays.
Archer’s Anchored Multiplex PCR (AMP™) technology overcomes this limitation, as it can enrich for novel fusions, while only requiring knowledge of one end of the fusion pair. At the heart of the AMP chemistry are unique Molecular Barcode (MBC) adapters, ligated to the 5′ ends of DNA fragments prior to amplification. The MBCs contain universal primer binding sites for PCR and a molecular barcode for identifying unique molecules. When combined with 3′ gene-specific primers, MBCs enable amplification of target regions with unknown 5′ ends.
“Tagging each molecule of input nucleic acid with a unique molecular barcode allows for de-duplication, error correction, and quantitative analysis, resulting in high sequencing consensus. With its low error rate and low limits of detection, AMP is revolutionizing the field of cancer genomics.”
In a proof-of-concept study, a single-tube 23-plex panel was designed to amplify the kinase domains of ALK, RET, ROS1, and MUSK genes by AMP. This enrichment strategy enabled identification of gene fusions with multiple partners and alternative splicing events in lung cancer, thyroid cancer, and glioblastoma specimens by NGS.
Over the last decade, the Biomarker/Translational Research Laboratory has focused on developing clinical genotyping and fluorescent in situ hybridization (FISH) assays for rapid personalized genomic testing.
“Initially, we analyzed the most prevalent hotspot mutations, about 160 in 25 cancer genes,” continued Dr. Borger. “However, this approach revealed mutations in only half of our patients. With the advent of NGS, we are able to sequence 190 exons in 39 cancer genes and obtain significantly richer genetic fingerprints, finding genetic aberrations in 92% of our cancer patients.”
Using multiplexed approaches, Dr. Borger’s team within the larger Center for Integrated Diagnostics (CID) program at MGH has established high-throughput genotyping service as an important component of routine care. While only a few susceptible molecular alterations may currently have a corresponding drug, the NGS-driven analysis may supply new information for inclusion of patients into ongoing clinical trials, or bank the result for future research and development.
“A significant impediment to discovery of clinically relevant genomic signatures is our current inability to interconnect the data,” explained Dr. Borger. “On the local level, we are striving to compile the data from clinical observations, including responses to therapy and genotyping. Globally, it is imperative that comprehensive public databases become available to the research community.”
This image, from the Massachusetts General Hospital Cancer Center, shows multicolor fluorescence in situ hybridization (FISH) analysis of cells from a patient with esophagogastric cancer. Remarkably, the FISH analysis revealed that co-amplification of the MET gene (red signal) and the EGFR gene (green signal) existed simultaneously in the same tumor cells. A chromosome 7 control probe is shown in blue.
Tumor profiling at MGH have already yielded significant discoveries. Dr. Borger’s lab, in collaboration with oncologists at the MGH Cancer Center, found significant correlations between mutations in the genes encoding the metabolic enzymes isocitrate dehydrogenase (IDH1 and IDH2) and certain types of cancers, such as cholangiocarcinoma and acute myelogenous leukemia (AML).
Historically, cancer signatures largely focus on signaling proteins. Discovery of a correlative metabolic enzyme offered a promise of diagnostics based on metabolic byproducts that may be easily identified in blood. Indeed, the metabolite 2-hydroxyglutarate accumulates to high levels in the tissues of patients carrying IDH1 and IDH2 mutations. They have reported that circulating 2-hydroxyglutarate as measured in the blood correlates with tumor burden, and could serve as an important surrogate marker of treatment response. …..
Researchers Uncover How ‘Silent’ Genetic Changes Drive Cancer
“Traditionally, it has been hard to use standard methods to quantify the amount of tRNA in the cell,” says Tavazoie. The lead authors of the article, Hani Goodarzi, formerly a postdoc in the lab and now a new assistant professor at UCSF, and research assistant Hoang Nguyen, devised and applied a new method that utilizes state-of-the-art genomic sequencing technology to measure the amount of tRNAs in different cell types.
The team chose to compare breast tissue from healthy individuals with tumor samples taken from breast cancer patients–including both primary tumors that had not spread from the breast to other body sites, and highly aggressive, metastatic tumors.
They found that the levels of two specific tRNAs were significantly higher in metastatic cells and metastatic tumors than in primary tumors that did not metastasize or healthy samples. “There are four different ways to encode for the protein building block arginine,” explains Tavazoie. “Yet only one of those–the tRNA that recognizes the codon CGG–was associated with increased metastasis.”
The tRNA that recognizes the codon GAA and encodes for a building block known as glutamic acid was also elevated in metastatic samples.
The team hypothesized that the elevated levels of these tRNAs may in fact drive metastasis. Working in mouse models of primary, non-metastatic tumors, the researchers increased the production of the tRNAs, and found that these cells became much more invasive and metastatic.
They also did the inverse experiment, with the anticipated results: reducing the levels of these tRNAs in metastatic cells decreased the incidence of metastases in the animals.
How do two tRNAs drive metastasis? The researchers teamed up with members of the Rockefeller University proteomics facility to see how protein expression changes in cells with elevated levels of these two tRNAs.
“We found global increases in many dozens of genes,” says Tavazoie, “so we analyzed their sequences and found that the majority of them had significantly increased numbers of these two specific codons.”
According to the researchers, two genes stood out among the list. Known as EXOSC2 and GRIPAP1, these genes were strongly and directly induced by elevated levels of the specific glutamic acid tRNA.
“When we mutated the GAA codons to GAG– a “silent” mutation because they both spell out the protein building block glutamic acid–we found that increasing the amount of tRNA no longer increased protein levels,” explains Tavazoie. These proteins were found to drive breast cancer metastasis.
The work challenges previous assumptions about how tRNAs function and suggests that tRNAs can modulate gene expression, according to the researchers. Tavazoie points out that “it is remarkable that within a single cell type, synonymous changes in genetic sequence can dramatically affect the levels of specific proteins, their transcripts, and the way a cell behaves.”
Testing Blood Metabolites Could Help Tailor Cancer Treatment
Scientists have found that measuring how cancer treatment affects the levels of metabolites – the building blocks of fats and proteins – can be used to assess whether the drug is hitting its intended target.
This new way of monitoring cancer therapy could speed up the development of new targeted drugs – which exploit specific genetic weaknesses in cancer cells – and help in tailoring treatment for patients.
Scientists at The Institute of Cancer Research, London, measured the levels of 180 blood markers in 41 patients with advanced cancers in a phase I clinical trial conducted with The Royal Marsden NHS Foundation Trust.
They found that investigating the mix of metabolic markers could accurately assess how cancers were responding to the targeted drug pictilisib.
Their study was funded by the Wellcome Trust, Cancer Research UK and the pharmaceutical company Roche, and is published in the journal Molecular Cancer Therapeutics.
Pictilisib is designed to specifically target a molecular pathway in cancer cells, called PI3 kinase, which has key a role in cell metabolism and is defective in a range of cancer types.
As cancers with PI3K defects grow, they can cause a decrease in the levels of metabolites in the bloodstream.
The new study is the first to show that blood metabolites are testable indicators of whether or not a new cancer treatment is hitting the correct target, both in preclinical mouse models and also in a trial of patients.
Using a sensitive technique called mass spectrometry, scientists at The Institute of Cancer Research (ICR) initially analysed the metabolite levels in the blood of mice with cancers that had defects in the PI3K pathway.
They found that the blood levels of 26 different metabolites, which were low prior to therapy, had risen considerably following treatment with pictilisib. Their findings indicated that the drug was hitting its target, and reversing the effects of the cancer on mouse metabolites.
Similarly, in humans the ICR researchers found that almost all of the metabolites – 22 out of the initial 26 – once again rose in response to pictilisib treatment, as seen in the mice.
Blood levels of the metabolites began to increase after a single dose of pictilisib, and were seen to drop again when treatment was stopped, suggesting that the effect was directly related to the drug treatment.
Metabolites vary naturally depending on the time of day or how much food a patient has eaten. But the researchers were able to provide the first strong evidence that despite this variation metabolites can be used to test if a drug is working, and could help guide decisions about treatment.
New Metabolic Pathway Reveals Aspirin-Like Compound’s Anti-Cancer Properties
Researchers at the Gladstone Institutes say they have found a new pathway by which salicylic acid, a key compound in the nonsteroidal anti-inflammatory drugs aspirin and diflunisal, stops inflammation and cancer.
In a study (“Salicylate, Diflunisal and Their Metabolites Inhibit CBP/p300 and Exhibit Anticancer Activity”) published in eLife, the investigators discovered that both salicylic acid and diflunisal suppress two key proteins that help control gene expression throughout the body. These sister proteins, p300 and CREB-binding protein (CBP), are epigenetic regulators that control the levels of proteins that cause inflammation or are involved in cell growth.
By inhibiting p300 and CBP, salicylic acid and diflunisal block the activation of these proteins and prevent cellular damage caused by inflammation. This study provides the first concrete demonstration that both p300 and CBP can be targeted by drugs and may have important clinical implications, according to Eric Verdin, M.D., associate director of the Gladstone Institute of Virology and Immunology .
“Salicylic acid is one of the oldest drugs on the planet, dating back to the Egyptians and the Greeks, but we’re still discovering new things about it,” he said. “Uncovering this pathway of inflammation that salicylic acid acts upon opens up a host of new clinical possibilities for these drugs.”
Earlier research conducted in the laboratory of co-author Stephen D. Nimer, M.D., director of Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, and a collaborator of Verdin’s, established a link between p300 and the leukemia-promoting protein AML1-ETO. In the current study, scientists at Gladstone and Sylvester worked together to test whether suppressing p300 with diflunisal would suppress leukemia growth in mice. As predicted, diflunisal stopped cancer progression and shrunk the tumors in the mouse model of leukemia. ……
Novel Protein Agent Targets Cancer and Host of Other Diseases
Researchers at Georgia State University have designed a new protein compound that can effectively target the cell surface receptor integrin v3, mutations in which have been linked to a number of diseases. Initial results using this new molecule show its potential as a therapeutic treatment for an array of illnesses, including cancer.
The novel protein molecule targets integrin v3 at a novel site that has not been targeted by other scientists. The researchers found that the molecule induces apoptosis, or programmed cell death, of cells that express integrin v3. This integrin has been a focus for drug development because abnormal expression of v3 is linked to the development and progression of various diseases.
“This integrin pair, v3, is not expressed in high levels in normal tissue,” explained senior study author Zhi-Ren Liu, Ph.D., professor in the department of biology at Georgia State. “In most cases, it’s associated with a number of different pathological conditions. Therefore, it constitutes a very good target for multiple disease treatment.”
“Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, which binds to integrin αvβ3 outside the classical ligand-binding site,” the authors wrote. “We show ProAgio induces apoptosis of integrin αvβ3-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin αvβ3.”
The findings from this study were published recently in Nature Communications in an article entitled “Rational Design of a Protein That Binds Integrin αvβ3 Outside the Ligand Binding Site.” …..
“We took a unique angle,” Dr. Lui noted. “We designed a protein that binds to a different site. Once the protein binds to the site, it directly triggers cell death. When we’re able to kill pathological cells, then we’re able to kill the disease.”
The investigators performed extensive cell and molecular testing that confirmed ProAgio interacts and binds well with integrin v3. Interestingly, they found that ProAgio induces apoptosis by recruiting caspase 8—an enzyme that plays an essential role in programmed cell death—to the cytoplasmic area of integrin v3. ProAgio was much more effective in inducing cell death than other agents tested.
Noncoding RNAs Not So Noncoding
Bits of the transcriptome once believed to function as RNA molecules are in fact translated into small proteins.
In 2002, a group of plant researchers studying legumes at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, discovered that a 679-nucleotide RNA believed to function in a noncoding capacity was in fact a protein-coding messenger RNA (mRNA).1 It had been classified as a long (or large) noncoding RNA (lncRNA) by virtue of being more than 200 nucleotides in length. The RNA, transcribed from a gene called early nodulin 40 (ENOD40), contained short open reading frames (ORFs)—putative protein-coding sequences bookended by start and stop codons—but the ORFs were so short that they had previously been overlooked. When the Cologne collaborators examined the RNA more closely, however, they found that two of the ORFs did indeed encode tiny peptides: one of 12 and one of 24 amino acids. Sampling the legumes confirmed that these micropeptides were made in the plant, where they interacted with a sucrose-synthesizing enzyme.
Five years later, another ORF-containing mRNA that had been posing as a lncRNA was discovered inDrosophila.2,3 After performing a screen of fly embryos to find lncRNAs, Yuji Kageyama, then of the National Institute for Basic Biology in Okazaki, Japan, suppressed each transcript’s expression. “Only one showed a clear phenotype,” says Kageyama, now at Kobe University. Because embryos missing this particular RNA lacked certain cuticle features, giving them the appearance of smooth rice grains, the researchers named the RNA “polished rice” (pri).
Turning his attention to how the RNA functioned, Kageyama thought he should first rule out the possibility that it encoded proteins. But he couldn’t. “We actually found it was a protein-coding gene,” he says. “It was an accident—we are RNA people!” The pri gene turned out to encode four tiny peptides—three of 11 amino acids and one of 32—that Kageyama and colleagues showed are important for activating a key developmental transcription factor.4
Since then, a handful of other lncRNAs have switched to the mRNA ranks after being found to harbor micropeptide-encoding short ORFs (sORFs)—those less than 300 nucleotides in length. And given the vast number of documented lncRNAs—most of which have no known function—the chance of finding others that contain micropeptide codes seems high.
Overlooked ORFs
From the late 1990s into the 21st century, as species after species had their genomes sequenced and deposited in databases, the search for novel genes and their associated mRNAs duly followed. With millions or even billions of nucleotides to sift through, researchers devised computational shortcuts to hunt for canonical gene and mRNA features, such as promoter regions, exon/intron splice sites, and, of course, ORFs.
ORFs can exist in practically any stretch of RNA sequence by chance, but many do not encode actual proteins. Because the chance that an ORF encodes a protein increases with its length, most ORF-finding algorithms had a size cut-off of 300 nucleotides—translating to 100 amino acids. This allowed researchers to “filter out garbage—that is, meaningless ORFs that exist randomly in RNAs,” says Eric Olsonof the University of Texas Southwestern Medical Center in Dallas.
Of course, by excluding all ORFs less than 300 nucleotides in length, such algorithms inevitably missed those encoding genuine small peptides. “I’m sure that the people who came up with [the cut-off] understood that this rule would have to miss anything that was shorter than 100 amino acids,” saysNicholas Ingolia of the University of California, Berkeley. “As people applied this rule more and more, they sort of lost track of that caveat.” Essentially, sORFs were thrown out with the computational trash and forgotten.
Aside from statistical practicality and human oversight, there were also technical reasons that contributed to sORFs and their encoded micropeptides being missed. Because of their small size, sORFs in model organisms such as mice, flies, and fish are less likely to be hit in random mutagenesis screens than larger ORFs, meaning their functions are less likely to be revealed. Also, many important proteins are identified based on their conservation across species, says Andrea Pauli of the Research Institute of Molecular Pathology in Vienna, but “the shorter [the ORF], the harder it gets to find and align this region to other genomes and to know that this is actually conserved.”
As for the proteins themselves, the standard practice of using electrophoresis to separate peptides by size often meant micropeptides would be lost, notes Doug Anderson, a postdoc in Olson’s lab. “A lot of times we run the smaller things off the bottom of our gels,” he says. Standard protein mass spectrometry was also problematic for identifying small peptides, says Gerben Menschaert of Ghent University in Belgium, because “there is a washout step in the protocol so that only larger proteins are retained.”
But as researchers take a deeper dive into the function of the thousands of lncRNAs believed to exist in genomes, they continue to uncover surprise micropeptides. In February 2014, for example, Pauli, then a postdoc in Alex Schier’s lab at Harvard University, discovered a hidden code in a zebrafish lncRNA. She had been hunting for lncRNAs involved in zebrafish development because “we hadn’t really anticipated that there would be any coding regions out there that had not been discovered—at least not something that is essential,” she says. But one lncRNA she identified actually encoded a 58-amino-acid micropeptide, which she called Toddler, that functioned as a signaling protein necessary for cell movements that shape the early embryo.5
Then, last year, Anderson and his colleagues reported another. Since joining Olson’s lab in 2010, Anderson had been searching for lncRNAs expressed in the heart and skeletal muscles of mouse embryos. He discovered a number of candidates, but one stood out for its high level of sequence conservation—suggesting to Anderson that it might have an important function. He was right, the RNA was important, but for a reason that neither Anderson nor Olson had considered: it was in fact an mRNA encoding a 46-amino-acid-long micropeptide.6
“When we zeroed in on the conserved region [of the gene], Doug found that it began with an ATG [start] codon and it terminated with a stop codon,” Olson says. “That’s when he looked at whether it might encode a peptide and found that indeed it did.” The researchers dubbed the peptide myoregulin, and found that it functioned as a critical calcium pump regulator for muscle relaxation.
With more and more overlooked peptides now being revealed, the big question is how many are left to be discovered. “Were there going to be dozens of [micropeptides]? Were there going to be hundreds, like there are hundreds of microRNAs?” says Ingolia. “We just didn’t know.”
Little things mean a lot. To any biologist, this time-worn maxim is old news. But it’s worth revisiting. As several articles in this issue of The Scientist illustrate, how researchers define and examine the “little things” does mean a lot.
Consider this month’s cover story, “Noncoding RNAs Not So Noncoding,” by TS correspondent Ruth Williams. Combing the human genome for open reading frames (ORFs), sequences bracketed by start and stop codons, yielded a protein-coding count somewhere in the neighborhood of 24,000. That left a lot of the genome relegated to the category of junk—or, later, to the tens of thousands of mostly mysterious long noncoding RNAs (lncRNAs). But because they had only been looking for ORFs that were 300 nucleotides or longer (i.e., coding for proteins at least 100 amino acids long), genome probers missed so-called short ORFs (sORFs), which encode small peptides. “Their diminutive size may have caused these peptides to be overlooked, their sORFs to be buried in statistical noise, and their RNAs to be miscategorized, but it does not prevent them from serving important, often essential functions, as the micropeptides characterized to date demonstrate,” writes Williams.
How little things work definitely informs another field of life science research: synthetic biology. As the functions of genes and gene networks are sussed out, bioengineers are using the information to design small, synthetic gene circuits that enable them to better understand natural networks. In “Synthetic Biology Comes into Its Own,” Richard Muscat summarizes the strides made by synthetic biologists over the last 15 years and offers an optimistic view of how such networks may be put to use in the future. And to prove him right, just as we go to press, a collaborative group led by one of syn bio’s founding fathers, MIT’s James Collins, has devised a paper-based test for Zika virus exposure that relies on a freeze-dried synthetic gene circuit that changes color upon detection of RNAs in the viral genome. The results are ready in a matter of hours, not the days or weeks current testing takes, and the test can distinguish Zika from dengue virus. “What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” commented one researcher about the test.
Moving around little things is the name of the game when it comes to delivering a package of drugs to a specific target or to operating on minuscule individual cells. Mini-scale delivery of biocompatible drug payloads often needs some kind of boost to overcome fluid forces or size restrictions that interfere with fine-scale manipulation. To that end, ingenious solutions that motorize delivery by harnessing osmotic changes, magnets, ultrasound, and even bacterial flagella are reviewed in “Making Micromotors Biocompatible.”
Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins αvβ3, αvβ5 and α5β1. It was developed by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive αvβ3 inhibitor (100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet receptor αIIbβ3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany).
This article describes the chemical development of Cilengitide, the biochemical background of its activity and a short review about the present clinical trials. The positive anti-angiogenic effects in cancer treatment can be further increased by combination with “classical” anti-cancer therapies. Several clinical trials in this direction are under investigation.
Integrins are heterodimeric receptors that are important for cell-cell and cell-extracellular matrix (ECM) interactions and are composed of one α and one β-subunit [1, 2]. These cell adhesion molecules act as transmembrane linkers between their extracellular ligands and the cytoskeleton, and modulate various signaling pathways essential in the biological functions of most cells. Integrins play a crucial role in processes such as cell migration, differentiation, and survival during embryogenesis, angiogenesis, wound healing, immune and non-immune defense mechanisms, hemostasis and oncogenic transformation [1]. The fact that many integrins are also linked with pathological conditions has converted them into very promising therapeutic targets [3]. In particular, integrins αvβ3, αvβ5 and α5β1 are involved in angiogenesis and metastasis of solid tumors, being excellent candidates for cancer therapy [4–7].
There are a number of different integrin subtypes which recognize and bind to the tripeptide sequence RGD (arginine, glycine, aspartic acid), which represents the most prominent recognition motif involved in cell adhesion. For example, the pro-angiogenic αvβ3 integrin binds various RGD-containing proteins, including fibronectin (Fn), fibrinogen (Fg), vitronectin (Vn) and osteopontin [8]. It is therefore not surprising that this integrin has been targeted for cancer therapy and that RGD-containing peptides and peptidomimetics have been designed and synthesized aiming to selectively inhibit this receptor [9, 10].
One classical strategy used in drug design is based on the knowledge about the structure of the receptor-binding pocket, preferably in complex with the natural ligand. However, this strategy, the so-called “rational structure-based design”, could not be applied in the field of integrin ligands since the first structures of integrin’s extracellular head groups were not described until 2001 for αvβ3 [11] (one year later, in 2002 the structure of this integrin in complex with Cilengitide was also reported [12]) and 2004 for αIIbβ3 [13]. Therefore, initial efforts in this field focused on a “ligand-oriented design”, which concentrated on optimizing RGD peptides by means of different chemical approaches in order to establish structure-activity relationships and identify suitable ligands.
We focused our interest in finding ligands for αvβ3 and based our approach on three chemical strategies pioneered in our group: 1) Reduction of the conformational space by cyclization; 2) Spatial screening of cyclic peptides; and 3)N-Methyl scan.
The combination of these strategies lead to the discovery of the cyclic peptidec(RGDf(NMe)V) in 1995. This peptide showed subnanomolar antagonistic activity for the αvβ3 receptor, nanomolar affinities for the closely related integrins αvβ5 and α5β1, and high selectivity towards the platelet receptor αIIbβ3. The peptide was patented together with Merck in 1997 (patent application submitted in 15.9.1995, opened in 20.3.1997) [14] and first presented with Merck’s agreement at the European Peptide Symposium in Edinburgh (September 1996) [15]. The synthesis and activity of this molecule was finally published in 1999 [16]. This peptide is now developed by Merck-Serono, (Darmstadt, Germany) under the name “Cilengitide” and has recently entered Phase III clinical trials for treating glioblastoma [17]. …..
The discovery 30 years ago of the RGD motif in Fn was a major breakthrough in science. This tripeptide sequence was also identified in other ECM proteins and was soon described as the most prominent recognition motif involved in cell adhesion. Extensive research in this direction allowed the description of a number of bidirectional proteins, the integrins, which were able to recognize and bind to the RGD sequence. Integrins are key players in the biological function of most cells and therefore the inhibition of RGD-mediated integrin-ECM interactions became an attractive target for the scientific community.
However, the lack of selectivity of linear RGD peptides represented a major pitfall which precluded any clinical application of RGD-based inhibitors. The control of the molecule’s conformation by cyclization and further spatial screening overcame these limitations, showing that it is possible to obtain privileged bioactive structures, which enhance the biological activity of linear peptides and significantly improve their receptor selectivity. Steric control imposed in RGD peptides together with their biological evaluation and extensive structural studies yielded the cyclic peptide c(RGDfV), the first small selective anti-angiogenic molecule described. N-Methylation of this cyclic peptide yielded the much potentc(RGDf(NMe)V), nowadays known as Cilengitide.
The fact that brain tumors, which are highly angiogenic, are more susceptible to the treatment with integrin antagonists, and the positive synergy observed for Cilengitide in combination with radio-chemotherapy in preclinical studies, encouraged subsequent clinical trials. Cilengitide is currently in phase III for GBM patients and in phase II for other types of cancers, with to date a promising therapeutic outcome. In addition, the absence of significant toxicity and excellent tolerance of this drug allows its combination with classical therapies such as RT or cytotoxic agents. The controlled phase III study CENTRIC was launched in 2008, with primary outcome measures due on September 2012. The results of this and other clinical studies are expected with great hope and interest.
Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
Integrins are heterodimeric cell surface receptors found in nearly all metazoan cell types, composed of non-covalently linked α and β subunits. In mammals, eighteen α-subunits and eight β-subunits have been identified to date 1. From this pool, 24 distinct heterodimer combinations have been observed in vivo that confer cell-to-cell and cell-to-ligand specificity relevant to the host cell and the environment in which it functions 2. Integrin-mediated interactions with the extracellular matrix (ECM) are required for the attachment, cytoskeletal organization, mechanosensing, migration, proliferation, differentiation and survival of cells in the context of a multitude of biological processes including fertilization, implantation and embryonic development, immune response, bone resorption and platelet aggregation. Integrins also function in pathological processes such as inflammation, wound healing, angiogenesis, and tumor metastasis. In addition, integrin binding has been identified as a means of viral entry into cells 3. ….
Combination of cilengitide and radiation therapy and temozolomide. The addition of cilengitide to radiotherapy and temozolomide based treatment regimens has shown promising preliminary results in ongoing Phase II trials in both newly diagnosed and progressive glioblastoma multiforme 139–140. In addition to the Phase II objectives sought, these trials are significant in that they represent progress that has made in determining tumor drug uptake and in identifying a subset of patients that may benefit from treatment. In a Phase II trial enrolling 52 patients with newly diagnosed glioblastoma multiforme receiving 500 mg cilengitide twice weekly during radiotherapy and in combination with temozolomide for 6 monthly cycles following radiotherapy, 69% achieved 6 months progression free survival compared to 54 % of patients receiving radiotherapy followed by temozolomide alone. The one-year overall survival was 67 and 62 % of patients for the cilengitide combination group and the radiotherapy and temozolomide group, respectively. Non-hematological grade 3-4 toxcities were limited, and included symptoms of fatigue, asthenia, anorexia, elevated liver function tests, deep vein thrombosis and pulmonary embolism in across a total of 5.7% of the patients. Grade 3-4 hematological malignancies were more common and included lymphopenia (53.8%), thrombocytopenia (13.4%) and neutropenia (9.6%). This trial is significant in the fact that is has provided the first evidence correlating a molecular biomarker with response to treatment. Decreased methylguanine methyltransferase (MGMT) expression was associated with favorable outcome. Patients harboring increased MGMT promoter methylation appeared to benefit more from combined treatment with cilengitide than did patients lacking promoter methylation. The significance of the MGMT promoter methylation in predicting response is likely due to inclusion of temozolomide in the treatment combination.
A similar Phase II study evaluating safety and differences in overall survival among newly diagnosed glioblastoma multiforme patients receiving radiation therapy combined with temozolomide and varying doses of cilengitide is nearing completion. Preliminary reports specify that initial safety run-in studies in 18 patients receiving doses 500, 1000 and 2000 mg cilengitide found no dose limiting toxicities. Subsequently 94 patients were randomized to receive standard therapy plus 500 or 2000 mg cilengitide. Median survival time in both cohorts was 18.9 months. At 12 months the overall survival was 79.5 % (89/112 patients).
In the last two decades great progress has been made in the discovery and development of integrin targeted therapeutics. Years of intense research into integrin function has provided an understanding of the potential applications for the treatment of disease. Advances in structural characterization of integrin-ligand interactions has proved beneficial in the design and development of potent, selective inhibitors for a number of integrins involved in platelet aggregation, inflammatory responses, angiongenesis, neovascularization and tumor growth.
The αIIbβ3 integrin antagonists were the first inhibitors to make their way into clinical use and have proven to be effective and safe drugs, contributing to the reduction of mortality and morbidity associated with acute coronary syndromes. Interestingly, the prolonged administration of small molecules targeting this integrin for long-term prevention of thrombosis related complications have not been successful, for reasons that are not yet fully understood. This suggests that modulating the intensity, duration and temporal aspects of integrin function may be more effective than simply shutting off integrin signaling in some instances. Further research into the dynamics of platelet activation and thrombosis formation may elucidate the mechanisms by which integrin activation is modulated.
The introduction of α4 targeted therapies held great promise for the treatment of inflammatory diseases. The development of Natalizumab greatly improved the quality of life for multiple sclerosis patients and those suffering with Crohn’s Disease compared to previous treatments, but the role in asthma related inflammation could not be validated. Unfortunately for MS and Crohn’s patients, immune surveillance in the central nervous system was also compromised as a direct effect α4β7 antagonism, with potentially lethal effects. Thus Natalizumab and related α4β7 targeting drugs are now limited to patients refractory to standard therapies. The design and development of α4β1 antagonists for the treatment of Crohn’s Disease may offer benefit with decreased risks. The involvement of these integrins in fetal development also raises concerns for widespread clinical use.
Integrin antagonists that target angiogenesis are progressing through clinical trials. Cilengitide has shown promising results for the treatment of glioblastomas and recurrent gliomas, cancers with notoriously low survival and cure rates. The greatest challenge facing the development of anti-angiogenic integrin targeted therapies is the overall lack of biomarkers by which to measure treatment efficacy.
Mapping the ligand-binding pocket of integrin α5β1 using a gain-of-function approach
Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by α5β1 is poorly understood. Here we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the recognition of RGD and synergy sites in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish α5 subunit with the corresponding regions of human α5 we show that blades 1-4 of the β-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the β-propeller domain. We find that the loop connecting blades 2 and 3 of the β-propeller (D3-A3 loop) contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human α5β1 supports an important function for D3-A3 loop residues Trp-157 and Ala-158 in the binding of antagonists. These results will aid the development of reagents that block α5β1 functions in vivo.
Structural Basis of Integrin Regulation and Signaling
Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cellpathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 Å couple to conformational change in ligand-binding sites and are linked to changes in α and β subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.
The immune system relies heavily on integrins for (a) adhesion during leukocyte trafficking from the bloodstream, migration within tissues, immune synapse formation, and phagocytosis; and (b) signaling during costimulation and cell polarization. Integrins are so named because they integrate the extracellular and intracellular environments by binding to ligands outside the cell and cytoskeletal components and signaling molecules inside the cell. Integrins are noncovalently associated heterodimeric cell surface adhesion molecules. In vertebrates, 18 α subunits and 8 β subunits form 24 known αβ pairs (Figure 1). This diversity in subunit composition contributes to diversity in ligand recognition, binding to cytoskeletal components and coupling to downstream signaling pathways. Immune cells express at least 10 members of the integrin family belonging to the β2, β7, and β1 subfamilies (Table 1). The β2 and β7 integrins are exclusively expressed on leukocytes, whereas the β1 integrins are expressed on a wide variety of cells throughout the body. Distribution and ligand-binding properties of the integrins on leukocytes are summarized in Table 1. For reviews, see References 1 and 2. Mutations that block expression of the β2 integrin subfamily lead to leukocyte adhesion deficiency, a disease associated with severe immunodeficiency (3).
As adhesion molecules, integrins are unique in that their adhesiveness can be dynamically regulated through a process termed inside-out signaling or priming. Thus, stimuli received by cell surface receptors for chemokines, cytokines, and foreign antigens initiate intracellular signals that impinge on integrin cytoplasmic domains and alter adhesiveness for extracellular ligands. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction (outside-in signaling). These dynamic properties of integrins are central to their proper function in the immune system. Indeed, mutations or small molecules that stabilize either the inactive state or the active adhesive state—and thereby block the adhesive dynamics of leukocyte integrins—inhibit leukocyte migration and normal immune responses.
Metabolomics: its Applications in Food and Nutrition Research
Reporter and Curator: Sudipta Saha, Ph.D.
Metabolomics is a relatively new field of “omics” research concerned with the high-throughput identification and quantification of small molecule (<1500 Da) metabolites in the metabolome. The metabolome is formally defined as the collection of all small molecule metabolites or chemicals that can be found in a cell, organ or organism. These small molecules can include a range of endogenous and exogenous chemical entities such as peptides, amino acids, nucleic acids, carbohydrates, organic acids, vitamins, polyphenols, alkaloids, minerals and just about any other chemical that can be used, ingested or synthesized by a given cell or organism.
Metabolomics is ideally positioned to be used in many areas of food science and nutrition research including food component analysis, food quality/authenticity assessment, food consumption monitoring and physiological monitoring in food intervention studies. However, the potential impact of metabolomics is still limited by two factors: (1) technology and (2) databases. In terms of instrumentation, it is clear that significant improvements need to be made to make metabolite detection and quantification technology more robust, automated and comprehensive. While promising advances have been made, current techniques are only capable of detecting perhaps 1/10th of the relevant metabolome. This expanded breadth and depth of coverage is particularly important in food and nutrition studies.
Many more reference spectral or chromatographic databases on metabolites, food components and phytochemicals need to be developed and made public. It is only through these databases that nutritionally relevant compounds can be routinely identified or quantified. Indeed a comprehensive effort, similar to that undertaken to annotate the human metabolome, needs to be made to complete and annotate the “food metabolome”. Similar efforts also need to be directed towards creating publicly accessible, comprehensive nutritional phenotype databases that include quantitative metabolomic (and other omic) data collected from diet-challenge or food intervention experiments. While these kinds of endeavours may take years to complete and cost millions of dollars, hopefully the food science community (and its funding agencies) will find a way of coordinating its activities to complete these efforts. Indeed, having public resource like a food metabolome database or a nutritional phenotype database could be as valuable to food scientists as GenBank has been to molecular biologists.
Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com
English: The citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle) or the Krebs cycle. Produced at WikiPathways. (Photo credit: Wikipedia)
Expanding the Genetic Alphabet and Linking the Genome to the Metabolome
Reporter& Curator: Larry Bernstein, MD, FCAP
Unlocking the diversity of genomic expression within tumorigenesis and “tailoring” of therapeutic options
1. Reshaping the DNA landscape between diseases and within diseases by the linking of DNA to treatments
In the NEW York Times of 9/24,2012 Gina Kolata reports on four types of breast cancer and the reshaping of breast cancer DNA treatment based on the findings of the genetically distinct types, which each have common “cluster” features that are driving many cancers. The discoveries were published online in the journal Nature on Sunday (9/23). The study is considered the first comprehensive genetic analysis of breast cancer and called a roadmap to future breast cancer treatments. I consider that if this is a landmark study in cancer genomics leading to personalized drug management of patients, it is also a fitting of the treatment to measurable “combinatorial feature sets” that tie into population biodiversity with respect to known conditions. The researchers caution that it will take years to establish transformative treatments, and this is clearly because in the genetic types, there are subsets that have a bearing on treatment “tailoring”. In addition, there is growing evidence that the Watson-Crick model of the gene is itself being modified by an expansion of the alphabet used to construct the DNA library, which itself will open opportunities to explain some of what has been considered junk DNA, and which may carry essential information with respect to metabolic pathways and pathway regulation. The breast cancer study is tied to the “Cancer Genome Atlas” Project, already reported. It is expected that this work will tie into building maps of genetic changes in common cancers, such as, breast, colon, and lung. What is not explicit I presume is a closely related concept, that the translational challenge is closely related to the suppression of key proteomic processes tied into manipulating the metabolome.
2. Fiddling with an expanded genetic alphabet – greater flexibility in design of treatment (pharmaneogenesis?)
Diagram of DNA polymerase extending a DNA strand and proof-reading. (Photo credit: Wikipedia)
A clear indication of this emerging remodeling of the genetic alphabet is a new
study led by scientists at The Scripps Research Institute appeared in the
June 3, 2012 issue of Nature Chemical Biology that indicates the genetic code as
we know it may be expanded to include synthetic and unnatural sequence pairing (Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought, Genome). They infer that the genetic instructions for living organisms
that is composed of four bases (C, G, A and T)— is open to unnatural letters. An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications. The implications of the application of this would further expand the translation of portions of DNA to new transciptional proteins that are heretofore unknown, but have metabolic relavence and therapeutic potential. The existence of such pairing in nature has been studied in Eukariotes for at least a decade, and may have a role in biodiversity. The investigators show how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases. This could as well be translated into human diversity, and human diseases.
The Romesberg laboratory collaborated on the new study and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G). It had been clear that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication, but that is not the case because other bonds can be in play.
The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion that resembles a ‘mispair.’ In test after test, the NaM-5SICS pair was efficiently replicable even though it appeared that the DNA polymerase didn’t recognize it. Their structural data showed that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication. NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium.
The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could be used to extend the DNA alphabet and that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been developed allowing for life based on other genetic systems.
3. Studies that consider a DNA triplet model that includes one or more NATURAL nucleosides and looks closely allied to the formation of the disulfide bond and oxidation reduction reaction.
This independent work is being conducted based on a similar concep. John Berger, founder of Triplex DNA has commented on this. He emphasizes Sulfur as the most important element for understanding evolution of metabolic pathways in the human transcriptome. It is a combination of sulfur 34 and sulphur 32 ATMU. S34 is element 16 + flourine, while S32 is element 16 + phosphorous. The cysteine-cystine bond is the bridge and controller between inorganic chemistry (flourine) and organic chemistry (phosphorous). He uses a dual spelling, using sulfphur to combine the two referring to the master catalyst of oxidation-reduction reactions. Various isotopic alleles (please note the duality principle which is natures most important pattern). Sulfphur is Methionine, S adenosylmethionine, cysteine, cystine, taurine, gluthionine, acetyl Coenzyme A, Biotin, Linoic acid, H2S, H2SO4, HSO3-, cytochromes, thioredoxin, ferredoxins, purple sulfphur anerobic bacteria prokaroytes, hydrocarbons, green sulfphur bacteria, garlic, penicillin and many antibiotics; hundreds of CSN drugs for parasites and fungi antagonists. These are but a few names which come to mind. It is at the heart of the Krebs cycle of oxidative phosphorylation, i.e. ATP. It is also a second pathway to purine metabolism and nucleic acids. It literally is the key enzymes between RNA and DNA, ie, SH thiol bond oxidized to SS (dna) cysteine through thioredoxins, ferredoxins, and nitrogenase. The immune system is founded upon sulfphur compounds and processes. Photosynthesis Fe4S4 to Fe2S3 absorbs the entire electromagnetic spectrum which is filtered by the Allen belt some 75 miles above earth. Look up chromatium vinosum or allochromatium species. There is reasonable evidence it is the first symbiotic species of sulfphur anerobic bacteria (Fe4S4) with high potential mvolts which drives photosynthesis while making glucose with H2S.
He envisions a sulfphur control map to automate human metabolism with exact timing sequences, at specific three dimensional coordinates on Bravais crystalline lattices. He proposes adding the inosine-xanthosine family to the current 5 nucleotide genetic code. Finally, he adds, the expanded genetic code is populated with “synthetic nucleosides and nucleotides” with all kinds of customized functional side groups, which often reshape nature’s allosteric and physiochemical properties. The inosine family is nature’s natural evolutionary partner with the adenosine and guanosine families in purine synthesis de novo, salvage, and catabolic degradation. Inosine has three major enzymes (IMPDH1,2&3 for purine ring closure, HPGRT for purine salvage, and xanthine oxidase and xanthine dehydrogenase.
English: DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule. This process is paramount to all life as we know it. (Photo credit: Wikipedia)
3. Nutritional regulation of gene expression, an essential role of sulfur, and metabolic control
Finally, the research carried out for decades by Yves Ingenbleek and the late Vernon Young warrants mention. According to their work, sulfur is again tagged as essential for health. Sulfur (S) is the seventh most abundant element measurable in human tissues and its provision is mainly insured by the intake of methionine (Met) found in plant and animal proteins. Met is endowed with unique functional properties as it controls the ribosomal initiation of protein syntheses, governs a myriad of major metabolic and catalytic activities and may be subjected to reversible redox processes contributing to safeguard protein integrity.
Consuming diets with inadequate amounts of methionine (Met) are characterized by overt or subclinical protein malnutrition, and it has serious morbid consequences. The result is reduction in size of their lean body mass (LBM), best identified by the serial measurement of plasma transthyretin (TTR), which is seen with unachieved replenishment (chronic malnutrition, strict veganism) or excessive losses (trauma, burns, inflammatory diseases). This status is accompanied by a rise in homocysteine, and a concomitant fall in methionine. The ratio of S to N is quite invariant, but dependent on source. The S:N ratio is typical 1:20 for plant sources and 1:14.5 for animal protein sources. The key enzyme involved with the control of Met in man is the enzyme cystathionine-b-synthase, which declines with inadequate dietary provision of S, and the loss is not compensated by cobalamine for CH3- transfer.
As a result of the disordered metabolic state from inadequate sulfur intake (the S:N ratio is lower in plants than in animals), the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. They both stimulate comparable remethylation reactions from homocysteine (Hcy), indicating that Met homeostasis benefits from high metabolic priority. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing reducing molecules implicated in the regulation of the 3 desulfuration pathways
4. The effect on accretion of LBM of protein malnutrition and/or the inflammatory state: in closer focus
Hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly and liver production of TTR integrates the dietary and stressful components of any disease spectrum. Thus we have a depletion of visceral transport proteins made by the liver and fat-free weight loss secondary to protein catabolism. This is most accurately reflected by TTR, which is a rapid turnover protein, but it is involved in transport and is essential for thyroid function (thyroxine-binding prealbumin) and tied to retinol-binding protein. Furthermore, protein accretion is dependent on a sulfonation reaction with 2 ATP. Consequently, Kwashiorkor is associated with thyroid goiter, as the pituitary-thyroid axis is a major sulfonation target. With this in mind, it is not surprising why TTR is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequaled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyper-homocysteinemic states.
Individuals submitted to N-restricted regimens are basically able to maintain N homeostasis until very late in the starvation processes. But the N balance study only provides an overall estimate of N gains and losses but fails to identify the tissue sites and specific interorgan fluxes involved. Using vastly improved methods the LBM has been measured in its components. The LBM of the reference man contains 98% of total body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass). The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g).
TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline with an accelerated decrease after 65 years. Sulfur Methylation (SM) undergoes a 15% reduction in size per decade, an involutive process. The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation or in critically ill patients whose muscle mass undergoes proteolysis.
5. The results of the events described are:
Declining generation of hydrogen sulfide (H2S) from enzymatic sources and in the non-enzymatic reduction of elemental S to H2S.
The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions.
Elemental S operates as co-factor of several (apo)enzymes critically involved in the control of oxidative processes.
Combination of protein and sulfur dietary deficiencies constitute a novel clinical entity threatening plant-eating population groups. They have a defective production of Cys, GSH and H2S reductants, explaining persistence of an oxidative burden.
6. The clinical entity increases the risk of developing:
cardiovascular diseases (CVD) and
stroke
in plant-eating populations regardless of Framingham criteria and vitamin-B status.
Met molecules supplied by dietary proteins are submitted to transmethylation processes resulting in the release of Hcy which:
either undergoes Hcy — Met RM pathways or
is committed to transsulfuration decay.
Impairment of CβS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. The data show that combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.
Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.
7. The dysfunctional metabolism in transitional cell transformation
A third development is also important and possibly related. The transition a cell goes through in becoming cancerous tends to be driven by changes to the cell’s DNA. But that is not the whole story. Large-scale techniques to the study of metabolic processes going on in cancer cells is being carried out at Oxford, UK in collaboration with Japanese workers. This thread will extend our insight into the metabolome. Otto Warburg, the pioneer in respiration studies, pointed out in the early 1900s that most cancer cells get the energy they need predominantly through a high utilization of glucose with lower respiration (the metabolic process that breaks down glucose to release energy). It helps the cancer cells deal with the low oxygen levels that tend to be present in a tumor. The tissue reverts to a metabolic profile of anaerobiosis. Studies of the genetic basis of cancer and dysfunctional metabolism in cancer cells are complementary. Tomoyoshi Soga’s large lab in Japan has been at the forefront of developing the technology for metabolomics research over the past couple of decades (metabolomics being the ugly-sounding term used to describe research that studies all metabolic processes at once, like genomics is the study of the entire genome).
Their results have led to the idea that some metabolic compounds, or metabolites, when they accumulate in cells, can cause changes to metabolic processes and set cells off on a path towards cancer. The collaborators have published a perspective article in the journal Frontiers in Molecular and Cellular Oncology that proposes fumarate as such an ‘oncometabolite’. Fumarate is a standard compound involved in cellular metabolism. The researchers summarize that shows how accumulation of fumarate when an enzyme goes wrong affects various biological pathways in the cell. It shifts the balance of metabolic processes and disrupts the cell in ways that could favor development of cancer. This is of particular interest because “fumarate” is the intermediate in the TCA cycle that is converted to malate.
Animation of the structure of a section of DNA. The bases lie horizontally between the two spiraling strands. (Photo credit: Wikipedia)
The Keio group is able to label glucose or glutamine, basic biological sources of fuel for cells, and track the pathways cells use to burn up the fuel. As these studies proceed, they could profile the metabolites in a cohort of tumor samples and matched normal tissue. This would produce a dataset of the concentrations of hundreds of different metabolites in each group. Statistical approaches could suggest which metabolic pathways were abnormal. These would then be the subject of experiments targeting the pathways to confirm the relationship between changed metabolism and uncontrolled growth of the cancer cells.
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