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Posts Tagged ‘Drug discovery’


Tumor Organoids Used to Speed Cancer Treatment

Reporter: Irina Robu, PhD

Collecting cancer cells from patients and growing them into 3-D mini tumors could make it possible to quickly screen large numbers of potential drugs for ultra-rare cancers. Preliminary success with a new high-speed, high-volume approach is already guiding treatment decisions for some patients with recurring hard-to-treat cancers.

A London-based team labelled how a “tumor-in-a-dish” approach positively forecasted drug responses in cancer patients who previously took part in clinical trials. That study was a major development in a new research area focused on “organoids” — tiny 3-D versions of the brain, gut, lung and other organs grown in the lab to probe basic biology or test drugs.

UCLA cancer biologist Alice Soragni and her colleagues developed a high-volume, automated method to rapidly study drug responses in tumor organoids grown from patient cells. By studying mini tumors grown on a plate with 96 tiny test tubes, her team can screen hundreds of compounds at once and classify promising candidates within a time frame that is therapeutically actionable. According to Dr. Soragni, the method seemed to work for various kinds of ovarian cancer. It was shown that the lab-grown organoids mimicked how tumors in the body look and behave. And even in cases when mini tumors had a hard time growing in a dish, scientists still acknowledged potential drug candidates.
Up to now, the UCLA team has produced organoids from 35 to 40 people with various types of sarcoma which will allow them to classify tumors that won’t respond to conventional therapy. This proves useful for people with recurrent metastases, where it’s not clear if we’re doing anything for their overall survival or giving them more toxicity.

Source

https://www.sciencenews.org/article/tumor-organoids-may-speed-cancer-treatment

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Targeting Cardio-Metabolic Diseases and Metabolomics in Drug Discovery – CHI’s 14th Annual Discovery On Target September 19-22, 2016 | Westin Boston Waterfront — Boston, MA

Reporter: Aviva Lev-Ari, PhD, RN

 

14th Annual Discovery On Target
September 19-22, 2016 | Westin Boston Waterfront — Boston, MA

2016 Prospectus Download | Current Sponsors | Current Exhibitors & Floorplan

Sponsorship Opportunities | 2015 Attendee List

Hello Aviva,

I wanted to inform you of the opportunity to meet with thought leaders at the 14th Annual Discovery On Target event, taking place September 19-22, 2016 in Boston attending the Targeting Cardio-Metabolic Diseases and Metabolomics in Drug Discovery conference programs. As a sponsor and/or exhibitor of this meeting, you have the opportunity to speak and network with 1,100+ attendees from 20+ countries composed of scientists, executives, directors, and managers from large biotech and pharmaceutical companies.

Delivering a sponsored presentation during the conference program is the most effective way to access even the hardest-to-reach decision makers from within your target market. This will increase your scientific presence and drive more qualified leads to your booth space, maximizing your ROI.

Please see the session topics, below:

This conference focuses on new cardiometabolic drug targets, mostly PCSK9 and the connections between cardiometabolic disease and liver metabolism, especially as manifested in a disease of the fatty liver, NASH (Non-Alcoholic SteatoHepatitis). 

Topics include:

  • New Cardiometabolic Drug Targets and PCSK9
  • NASH: Non-Alcohlic Steatohepatitis and Cardiometabolism

This conference will emphasize presentations that analyze metabolomics data in a larger context of cellular functioning or disease states. A few introductory type presentations will highlight the current state of the field and its major technologies.

Topics include:

  • Metabolomics Overview and Technologies
  • Disease-Focused Research Stemming from the Metabolomic Analysis
  • Cancer Metabolism

Opportunities are available for sponsored presentations during the conference agenda, One-on-One Meetings, and exhibit opportunities. Act now, as priority placement is given to companies who sign on early. We can customize a sponsorship package to meet your company’s needs and reach your target audience. Thank you for your time and I look forward to hearing from you!

Kind regards,

Jon Stroup

Senior Manager, Business Development
T: 781.972.5483
F: 781.972.5452
E: jstroup@cambridgeinnovationinstitute.com
W: DiscoveryOnTarget.com

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SPONSORSHIP & EXHIBIT INFORMATION

2016 Prospectus Now Available!

PREMIER SPONSOR

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From: Jon Stroup <sales2@healthtech.com>

Date: Monday, June 13, 2016 at 2:40 PM

To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>

Subject: Targeting Cardio-Metabolic Diseases & Metabolomics in Drug Discovery

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Electronic Proceedings for 10th US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

 

Curator: Aviva Lev-Ari, PhD, RN

 

LIVE — 9AM-noon US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

https://pharmaceuticalintelligence.com/2016/06/02/live-9am-noon-us-india-biopharma-healthcare-summit-june-2-2016-marriott-cambridge-ma/

 

LIVE 11:45AM – 2:40PM US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

https://pharmaceuticalintelligence.com/2016/06/02/1145am-240pm-us-india-biopharma-healthcare-summit-june-2-2016-marriott-cambridge-ma/

 

LIVE 3:15PM – 5:00PM US-India BioPharma & Healthcare Summit, June 2, 2016, Marriott Cambridge, MA

https://pharmaceuticalintelligence.com/2016/06/02/315pm-500pm-us-india-biopharma-healthcare-summit-june-2-2016-marriott-cambridge-ma/

 

 

 

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Gene Editing with CRISPR gets Crisper

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

 

CRISPR Moves from Butchery to Surgery   

More Genomes Are Going Under the CRISPR Knife, So Surgical Standards Are Rising

http://www.genengnews.com/gen-articles/crispr-moves-from-butchery-to-surgery/5759/

  • The Dharmacon subsidary of GE Healthcare provides the Edit-R Lentiviral Gene Engineering platform. It is based on the natural S. pyrogenes system, but unlike that system, which uses a single guide RNA (sgRNA), the platform uses two component RNAs, a gene-specific CRISPR RNA (crRNA) and a universal trans-activating crRNA (tracrRNA). Once hybridized to the universal tracrRNA (blue), the crRNA (green) directs the Cas9 nuclease to a specific genomic region to induce a double- strand break.

    Scientists recently convened at the CRISPR Precision Gene Editing Congress, held in Boston, to discuss the new technology. As with any new technique, scientists have discovered that CRISPR comes with its own set of challenges, and the Congress focused its discussion around improving specificity, efficiency, and delivery.

    In the naturally occurring system, CRISPR-Cas9 works like a self-vaccination in the bacterial immune system by targeting and cleaving viral DNA sequences stored from previous encounters with invading phages. The endogenous system uses two RNA elements, CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA), which come together and guide the Cas9 nuclease to the target DNA.

    Early publications that demonstrated CRISPR gene editing in mammalian cells combined the crRNA and tracrRNA sequences to form one long transcript called asingle-guide RNA (sgRNA). However, an alternative approach is being explored by scientists at the Dharmacon subsidiary of GE Healthcare. These scientists have a system that mimics the endogenous system through a synthetic two-component approach thatpreserves individual crRNA and tracrRNA. The tracrRNA is universal to any gene target or species; the crRNA contains the information needed to target the gene of interest.

    Predesigned Guide RNAs

    In contrast to sgRNAs, which are generated through either in vitro transcription of a DNA template or a plasmid-based expression system, synthetic crRNA and tracrRNA eliminate the need for additional cloning and purification steps. The efficacy of guide RNA (gRNA), whether delivered as a sgRNA or individual crRNA and tracrRNA, depends not only on DNA binding, but also on the generation of an indel that will deliver the coup de grâce to gene function.

    “Almost all of the gRNAs were able to create a break in genomic DNA,” said Louise Baskin, senior product manager at Dharmacon. “But there was a very wide range in efficiency and in creating functional protein knock-outs.”

    To remove the guesswork from gRNA design, Dharmacon developed an algorithm to predict gene knockout efficiency using wet-lab data. They also incorporated specificity as a component of their algorithm, using a much more comprehensive alignment tool to predict potential off-target effects caused by mismatches and bulges often missed by other alignment tools. Customers can enter their target gene to access predesigned gRNAs as either two-component RNAs or lentiviral sgRNA vectors for multiple applications.

    “We put time and effort into our algorithm to ensure that our guide RNAs are not only functional but also highly specific,” asserts Baskin. “As a result, customers don’t have to do any design work.”

    Donor DNA Formats

    http://www.genengnews.com/Media/images/Article/thumb_MilliporeSigma_CRISPR3120824917.jpg
    MilliporeSigma’s CRISPR Epigenetic Activator is based on fusion of a nuclease-deficient Cas9 (dCas9) to the catalytic histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300. This technology allows researchers to target specific DNA regions or gene sequences. Researchers can localize epigenetic changes to their target of interest and see the effects of those changes in gene expression.

    Knockout experiments are a powerful tool for analyzing gene function. However, for researchers who want to introduce DNA into the genome, guide design, donor DNA selection, and Cas9 activity are paramount to successful DNA integration.MilliporeSigma offers two formats for donor DNA: double-stranded DNA (dsDNA) plasmids and single-stranded DNA (ssDNA) oligonucleotides. The most appropriate format depends on cell type and length of the donor DNA. “There are some cell types that have immune responses to dsDNA,” said Gregory Davis, Ph.D., R&D manager, MilliporeSigma.

  • The ssDNA format can save researchers time and money, but it has a limited carrying capacity of approximately 120 base pairs.In addition to selecting an appropriate donor DNA format, controlling where, how, and when the Cas9 enzyme cuts can affect gene-editing efficiency. Scientists are playing tug-of-war, trying to pull cells toward the preferred homology-directed repair (HDR) and away from the less favored nonhomologous end joining (NHEJ) repair mechanism.One method to achieve this modifies the Cas9 enzyme to generate a nickase that cuts only one DNA strand instead of creating a double-strand break. Accordingly, MilliporeSigma has created a Cas9 paired-nickase system that promotes HDR, while also limiting off-target effects and increasing the number of sequences available for site-dependent gene modifications, such as disease-associated single nucleotide polymorphisms (SNPs).“The best thing you can do is to cut as close to the SNP as possible,” advised Dr. Davis. “As you move the double-stranded break away from the site of mutation you get an exponential drop in the frequency of recombination.”

 

  • Ribonucleo-protein Complexes

    Another strategy to improve gene-editing efficiency, developed by Thermo Fisher, involves combining purified Cas9 protein with gRNA to generate a stable ribonucleoprotein (RNP) complex. In contrast to plasmid- or mRNA-based formats, which require transcription and/or translation, the Cas9 RNP complex cuts DNA immediately after entering the cell. Rapid clearance of the complex from the cell helps to minimize off-target effects, and, unlike a viral vector, the transient complex does not introduce foreign DNA sequences into the genome.

    To deliver their Cas9 RNP complex to cells, Thermo Fisher has developed a lipofectamine transfection reagent called CRISPRMAX. “We went back to the drawing board with our delivery, screened a bunch of components, and got a brand-new, fully  optimized lipid nanoparticle formulation,” explained Jon Chesnut, Ph.D., the company’s senior director of synthetic biology R&D. “The formulation is specifically designed for delivering the RNP to cells more efficiently.”

    Besides the reagent and the formulation, Thermo Fisher has also developed a range of gene-editing tools. For example, it has introduced the Neon® transfection system for delivering DNA, RNA, or protein into cells via electroporation. Dr. Chesnut emphasized the company’s focus on simplifying complex workflows by optimizing protocols and pairing everything with the appropriate up- and downstream reagents.

From Mammalian Cells to Microbes

One of the first sources of CRISPR technology was the Feng Zhang laboratory at the Broad Institute, which counted among its first licensees a company called GenScript. This company offers a gene-editing service called GenCRISPR™ to establish mammalian cell lines with CRISPR-derived gene knockouts.

“There are a lot of challenges with mammalian cells, and each cell line has its own set of issues,” said Laura Geuss, a marketing specialist at GenScript. “We try to offer a variety of packages that can help customers who have difficult-to-work-with cells.” These packages include both viral-based and transient transfection techniques.

However, the most distinctive service offered by GenScript is its microbial genome-editing service for bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). The company’s strategy for gene editing in bacteria can enable seamless knockins, knockouts, or gene replacements by combining CRISPR with lambda red recombineering. Traditionally one of the most effective methods for gene editing in microbes, recombineering allows editing without restriction enzymes through in vivo homologous recombination mediated by a phage-based recombination system such as lambda red.

On its own, lambda red technology cannot target multiple genes, but when paired with CRISPR, it allows the editing of multiple genes with greater efficiency than is possible with CRISPR alone, as the lambda red proteins help repair double-strand breaks in E. coli. The ability to knockout different gene combinations makes Genscript’s microbial editing service particularly well suited for the optimization of metabolic pathways.

Pooled and Arrayed Library Strategies

Scientists are using CRISPR technology for applications such as metabolic engineering and drug development. Yet another application area benefitting from CRISPR technology is cancer research. Here, the use of pooled CRISPR libraries is becoming commonplace. Pooled CRISPR libraries can help detect mutations that affect drug resistance, and they can aid in patient stratification and clinical trial design.

Pooled screening uses proliferation or viability as a phenotype to assess how genetic alterations, resulting from the application of a pooled CRISPR library, affect cell growth and death in the presence of a therapeutic compound. The enrichment or depletion of different gRNA populations is quantified using deep sequencing to identify the genomic edits that result in changes to cell viability.

MilliporeSigma provides pooled CRISPR libraries ranging from the whole human genome to smaller custom pools for these gene-function experiments. For pharmaceutical and biotech companies, Horizon Discovery offers a pooled screening service, ResponderSCREEN, which provides a whole-genome pooled screen to identify genes that confer sensitivity or resistance to a compound. This service is comprehensive, taking clients from experimental design all the way through to suggestions for follow-up studies.

Horizon Discovery maintains a Research Biotech business unit that is focused on target discovery and enabling translational medicine in oncology. “Our internal backbone gives us the ability to provide expert advice demonstrated by results,” said Jon Moore, Ph.D., the company’s CSO.

In contrast to a pooled screen, where thousands of gRNA are combined in one tube, an arrayed screen applies one gRNA per well, removing the need for deep sequencing and broadening the options for different endpoint assays. To establish and distribute a whole-genome arrayed lentiviral CRISPR library, MilliporeSigma partnered with the Wellcome Trust Sanger Institute. “This is the first and only arrayed CRISPR library in the world,” declared Shawn Shafer, Ph.D., functional genomics market segment manager, MilliporeSigma. “We were really proud to partner with Sanger on this.”

Pooled and arrayed screens are powerful tools for studying gene function. The appropriate platform for an experiment, however, will be determined by the desired endpoint assay.

Detection and Quantification of Edits

 

The QX200 Droplet Digital PCR System from Bio-Rad Laboratories can provide researchers with an absolute measure of target DNA molecules for EvaGreen or probe-based digital PCR applications. The system, which can provide rapid, low-cost, ultra-sensitive quantification of both NHEJ- and HDR-editing events, consists of two instruments, the QX200 Droplet Generator and the QX200 Droplet Reader, and their associated consumables.

Finally, one last challenge for CRISPR lies in the detection and quantification of changes made to the genome post-editing. Conventional methods for detecting these alterations include gel methods and next-generation sequencing. While gel methods lack sensitivity and scalability, next-generation sequencing is costly and requires intensive bioinformatics.

To address this gap, Bio-Rad Laboratories developed a set of assay strategies to enable sensitive and precise edit detection with its Droplet Digital PCR (ddPCR) technology. The platform is designed to enable absolute quantification of nucleic acids with high sensitivity, high precision, and short turnaround time through massive droplet partitioning of samples.

Using a validated assay, a typical ddPCR experiment takes about five to six hours to complete. The ddPCR platform enables detection of rare mutations, and publications have reported detection of precise edits at a frequency of <0.05%, and of NHEJ-derived indels at a frequency as low as 0.1%. In addition to quantifying precise edits, indels, and computationally predicted off-target mutations, ddPCR can also be used to characterize the consequences of edits at the RNA level.

According to a recently published Science paper, the laboratory of Charles A. Gersbach, Ph.D., at Duke University used ddPCR in a study of muscle function in a mouse model of Duchenne muscular dystrophy. Specifically, ddPCR was used to assess the efficiency of CRISPR-Cas9 in removing the mutated exon 23 from the dystrophin gene. (Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene and significant enhancement of muscle force.)

Quantitative ddPCR showed that exon 23 was deleted in ~2% of all alleles from the whole-muscle lysate. Further ddPCR studies found that 59% of mRNA transcripts reflected the deletion.

“There’s an overarching idea that the genome-editing field is moving extremely quickly, and for good reason,” asserted Jennifer Berman, Ph.D., staff scientist, Bio-Rad Laboratories. “There’s a lot of exciting work to be done, but detection and quantification of edits can be a bottleneck for researchers.”

The gene-editing field is moving quickly, and new innovations are finding their way into the laboratory as researchers lay the foundation for precise, well-controlled gene editing with CRISPR.

 

Are Current Cancer Drug Discovery Methods Flawed?

GEN May 3, 2016   http://www.genengnews.com/gen-news-highlights/are-current-cancer-drug-discovery-methods-flawed/81252682/

 

Researchers utilized a systems biology approach to develop new methods to assess drug sensitivity in cells. [The Institute for Systems Biology]

Understanding how cells respond and proliferate in the presence of anticancer compounds has been the foundation of drug discovery ideology for decades. Now, a new study from scientists at Vanderbilt University casts significant suspicion on the primary method used to test compounds for anticancer activity in cells—instilling doubt on methods employed by the entire scientific enterprise and pharmaceutical industry to discover new cancer drugs.

“More than 90% of candidate cancer drugs fail in late-stage clinical trials, costing hundreds of millions of dollars,” explained co-senior author Vito Quaranta, M.D., director of the Quantitative Systems Biology Center at Vanderbilt. “The flawed in vitro drug discovery metric may not be the only responsible factor, but it may be worth pursuing an estimate of its impact.”

The Vanderbilt investigators have developed what they believe to be a new metric for evaluating a compound’s effect on cell proliferation—called the DIP (drug-induced proliferation) rate—that overcomes the flawed bias in the traditional method.

The findings from this study were published recently in Nature Methods in an article entitled “An Unbiased Metric of Antiproliferative Drug Effect In Vitro.”

For more than three decades, researchers have evaluated the ability of a compound to kill cells by adding the compound in vitro and counting how many cells are alive after 72 hours. Yet, proliferation assays that measure cell number at a single time point don’t take into account the bias introduced by exponential cell proliferation, even in the presence of the drug.

“Cells are not uniform, they all proliferate exponentially, but at different rates,” Dr. Quaranta noted. “At 72 hours, some cells will have doubled three times and others will not have doubled at all.”

Dr. Quaranta added that drugs don’t all behave the same way on every cell line—for example, a drug might have an immediate effect on one cell line and a delayed effect on another.

The research team decided to take a systems biology approach, a mixture of experimentation and mathematical modeling, to demonstrate the time-dependent bias in static proliferation assays and to develop the time-independent DIP rate metric.

“Systems biology is what really makes the difference here,” Dr. Quaranta remarked. “It’s about understanding cells—and life—as dynamic systems.”This new study is of particular importance in light of recent international efforts to generate data sets that include the responses of thousands of cell lines to hundreds of compounds. Using the

  • Cancer Cell Line Encyclopedia (CCLE) and
  • Genomics of Drug Sensitivity in Cancer (GDSC) databases

will allow drug discovery scientists to include drug response data along with genomic and proteomic data that detail each cell line’s molecular makeup.

“The idea is to look for statistical correlations—these particular cell lines with this particular makeup are sensitive to these types of compounds—to use these large databases as discovery tools for new therapeutic targets in cancer,” Dr. Quaranta stated. “If the metric by which you’ve evaluated the drug sensitivity of the cells is wrong, your statistical correlations are basically no good.”

The Vanderbilt team evaluated the responses from four different melanoma cell lines to the drug vemurafenib, currently used to treat melanoma, with the standard metric—used for the CCLE and GDSC databases—and with the DIP rate. In one cell line, they found a glaring disagreement between the two metrics.

“The static metric says that the cell line is very sensitive to vemurafenib. However, our analysis shows this is not the case,” said co-lead study author Leonard Harris, Ph.D., a systems biology postdoctoral fellow at Vanderbilt. “A brief period of drug sensitivity, quickly followed by rebound, fools the static metric, but not the DIP rate.”

Dr. Quaranta added that the findings “suggest we should expect melanoma tumors treated with this drug to come back, and that’s what has happened, puzzling investigators. DIP rate analyses may help solve this conundrum, leading to better treatment strategies.”

The researchers noted that using the DIP rate is possible because of advances in automation, robotics, microscopy, and image processing. Moreover, the DIP rate metric offers another advantage—it can reveal which drugs are truly cytotoxic (cell killing), rather than merely cytostatic (cell growth inhibiting). Although cytostatic drugs may initially have promising therapeutic effects, they may leave tumor cells alive that then have the potential to cause the cancer to recur.

The Vanderbilt team is currently in the process of identifying commercial entities that can further refine the software and make it widely available to the research community to inform drug discovery.

 

An unbiased metric of antiproliferative drug effect in vitro

Leonard A HarrisPeter L FrickShawn P GarbettKeisha N HardemanB Bishal PaudelCarlos F LopezVito Quaranta & Darren R Tyson
Nature Methods 2 May (2016)
                 doi:10.1038/nmeth.3852

In vitro cell proliferation assays are widely used in pharmacology, molecular biology, and drug discovery. Using theoretical modeling and experimentation, we show that current metrics of antiproliferative small molecule effect suffer from time-dependent bias, leading to inaccurate assessments of parameters such as drug potency and efficacy. We propose the drug-induced proliferation (DIP) rate, the slope of the line on a plot of cell population doublings versus time, as an alternative, time-independent metric.

  1. Zuber, J. et al. Nat. Biotechnol. 29, 7983 (2011).
  2. Berns, K. et al. Nature 428, 431437 (2004).
  3. Bonnans, C., Chou, J. & Werb, Z. Nat. Rev. Mol. Cell Biol. 15, 786801 (2014).
  4. Garnett, M.J. et al. Nature 483, 570575 (2012)

 

Mapping Traits to Genes with CRISPR

Researchers develop a technique to direct chromosome recombination with CRISPR/Cas9, allowing high-resolution genetic mapping of phenotypic traits in yeast.

By Catherine Offord | May 5, 2016

http://www.the-scientist.com/?articles.view/articleNo/46029/title/Mapping-Traits-to-Genes-with-CRISPR

 

Researchers used CRISPR/Cas9 to make a targeted double-strand break (DSB) in one arm of a yeast chromosome labeled with a green fluorescent protein (GFP) gene. A within-cell mechanism called homologous repair (HR) mends the broken arm using its homolog, resulting in a recombined region from the site of the break to the chromosome tip. When this cell divides by mitosis, each daughter cell will contain a homozygous section in an outcome known as “loss of heterozygosity” (LOH). One of the daughter cells is detectable because, due to complete loss of the GFP gene, it will no longer be fluorescent.REPRINTED WITH PERMISSION FROM M.J. SADHU ET AL., SCIENCE

When mapping phenotypic traits to specific loci, scientists typically rely on the natural recombination of chromosomes during meiotic cell division in order to infer the positions of responsible genes. But recombination events vary with species and chromosome region, giving researchers little control over which areas of the genome are shuffled. Now, a team at the University of California, Los Angeles (UCLA), has found a way around these problems by using CRISPR/Cas9 to direct targeted recombination events during mitotic cell division in yeast. The team described its technique today (May 5) in Science.

“Current methods rely on events that happen naturally during meiosis,” explained study coauthor Leonid Kruglyak of UCLA. “Whatever rate those events occur at, you’re kind of stuck with. Our idea was that using CRISPR, we can generate those events at will, exactly where we want them, in large numbers, and in a way that’s easy for us to pull out the cells in which they happened.”

Generally, researchers use coinheritance of a trait of interest with specific genetic markers—whose positions are known—to figure out what part of the genome is responsible for a given phenotype. But the procedure often requires impractically large numbers of progeny or generations to observe the few cases in which coinheritance happens to be disrupted informatively. What’s more, the resolution of mapping is limited by the length of the smallest sequence shuffled by recombination—and that sequence could include several genes or gene variants.

“Once you get down to that minimal region, you’re done,” said Kruglyak. “You need to switch to other methods to test every gene and every variant in that region, and that can be anywhere from challenging to impossible.”

But programmable, DNA-cutting champion CRISPR/Cas9 offered an alternative. During mitotic—rather than meiotic—cell division, rare, double-strand breaks in one arm of a chromosome preparing to split are sometimes repaired by a mechanism called homologous recombination. This mechanism uses the other chromosome in the homologous pair to replace the sequence from the break down to the end of the broken arm. Normally, such mitotic recombination happens so rarely as to be impractical for mapping purposes. With CRISPR/Cas9, however, the researchers found that they could direct double-strand breaks to any locus along a chromosome of interest (provided it was heterozygous—to ensure that only one of the chromosomes would be cut), thus controlling the sites of recombination.

Combining this technique with a signal of recombination success, such as a green fluorescent protein (GFP) gene at the tip of one chromosome in the pair, allowed the researchers to pick out cells in which recombination had occurred: if the technique failed, both daughter cells produced by mitotic division would be heterozygous, with one copy of the signal gene each. But if it succeeded, one cell would end up with two copies, and the other cell with none—an outcome called loss of heterozygosity.

“If we get loss of heterozygosity . . . half the cells derived after that loss of heterozygosity event won’t have GFP anymore,” study coauthor Meru Sadhu of UCLA explained. “We search for these cells that don’t have GFP out of the general population of cells.” If these non-fluorescent cells with loss of heterozygosity have the same phenotype as the parent for a trait of interest, then CRISPR/Cas9-targeted recombination missed the responsible gene. If the phenotype is affected, however, then the trait must be linked to a locus in the recombined, now-homozygous region, somewhere between the cut site and the GFP gene.

By systematically making cuts using CRISPR/Cas9 along chromosomes in a hybrid, diploid strain ofSaccharomyces cerevisiae yeast, picking out non-fluorescent cells, and then observing the phenotype, the UCLA team demonstrated that it could rapidly identify the phenotypic contribution of specific gene variants. “We can simply walk along the chromosome and at every [variant] position we can ask, does it matter for the trait we’re studying?” explained Kruglyak.

For example, the team showed that manganese sensitivity—a well-defined phenotypic trait in lab yeast—could be pinpointed using this method to a single nucleotide polymorphism (SNP) in a gene encoding the Pmr1 protein (a manganese transporter).

Jason Moffat, a molecular geneticist at the University of Toronto who was not involved in the work, toldThe Scientist that researchers had “dreamed about” exploiting these sorts of mechanisms for mapping purposes, but without CRISPR, such techniques were previously out of reach. Until now, “it hasn’t been so easy to actually make double-stranded breaks on one copy of a pair of chromosomes, and then follow loss of heterozygosity in mitosis,” he said, adding that he hopes to see the approach translated into human cell lines.

Applying the technique beyond yeast will be important, agreed cell and developmental biologist Ethan Bier of the University of California, San Diego, because chromosomal repair varies among organisms. “In yeast, they absolutely demonstrate the power of [this method],” he said. “We’ll just have to see how the technology develops in other systems that are going to be far less suited to the technology than yeast. . . . I would like to see it implemented in another system to show that they can get the same oomph out of it in, say, mammalian somatic cells.”

Kruglyak told The Scientist that work in higher organisms, though planned, is still in early stages; currently, his team is working to apply the technique to map loci responsible for trait differences between—rather than within—yeast species.

“We have a much poorer understanding of the differences across species,” Sadhu explained. “Except for a few specific examples, we’re pretty much in the dark there.”

M.J. Sadhu, “CRISPR-directed mitotic recombination enables genetic mapping without crosses,” Science, doi:10.1126/science.aaf5124, 2016.

 

CRISPR-directed mitotic recombination enables genetic mapping without crosses

Meru J Sadhu, Joshua S Bloom, Laura Day, Leonid Kruglyak

Thank you, David, for the kind words and comments. We agree that the most immediate applications of the CRISPR-based recombination mapping will be in unicellular organisms and cell culture. We also think the method holds a lot of promise for research in multicellular organisms, although we did not mean to imply that it “will be an efficient mapping method for all multicellular organisms”. Every organism will have its own set of constraints as well as experimental tools that will be relevant when adapting a new technique. To best help experts working on these organisms, here are our thoughts on your questions.

You asked about mutagenesis during recombination. We Sanger sequenced 72 of our LOH lines at the recombination site and did not observe any mutations, as described in the supplementary materials. We expect the absence of mutagenesis is because we targeted heterozygous sites where the untargeted allele did not have a usable PAM site; thus, following LOH, the targeted site is no longer present and cutting stops. In your experiments you targeted sites that were homozygous; thus, following recombination, the CRISPR target site persisted, and continued cutting ultimately led to repair by NHEJ and mutagenesis.

As to the more general question of the optimal mapping strategies in different organisms, they will depend on the ease of generating and screening for editing events, the cost and logistics of maintaining and typing many lines, and generation time, among other factors. It sounds like in Drosophila today, your related approach of generating markers with CRISPR, and then enriching for natural recombination events that separate them, is preferable. In yeast, we’ve found the opposite to be the case. As you note, even in Drosophila, our approach may be preferable for regions with low or highly non-uniform recombination rates.

Finally, mapping in sterile interspecies hybrids should be straightforward for unicellular hybrids (of which there are many examples) and for cells cultured from hybrid animals or plants. For studies in hybrid multicellular organisms, we agree that driving mitotic recombination in the early embryo may be the most promising approach. Chimeric individuals with mitotic clones will be sufficient for many traits. Depending on the system, it may in fact be possible to generate diploid individuals with uniform LOH genotype, but this is certainly beyond the scope of our paper. The calculation of the number of lines assumes that the mapping is done in a single step; as you note in your earlier comment, mapping sequentially can reduce this number dramatically.

This is a lovely method and should find wide applicability in many settings, especially for microorganisms and cell lines. However, it is not clear that this approach will be, as implied by the discussion, an efficient mapping method for all multicellular organisms. I have performed similar experiments in Drosophila, focused on meiotic recombination, on a much smaller scale, and found that CRISPR-Cas9 can indeed generate targeted recombination at gRNA target sites. In every case I tested, I found that the recombination event was associated with a deletion at the gRNA site, which is probably unimportant for most mapping efforts, but may be a concern in some specific cases, for example for clinical applications. It would be interesting to know how often mutations occurred at the targeted gRNA site in this study.

The wider issue, however, is whether CRISPR-mediated recombination will be more efficient than other methods of mapping. After careful consideration of all the costs and the time involved in each of the steps for Drosophila, we have decided that targeted meiotic recombination using flanking visible markers will be, in most cases, considerably more efficient than CRISPR-mediated recombination. This is mainly due to the large expense of injecting embryos and the extensive effort and time required to screen injected animals for appropriate events. It is both cheaper and faster to generate markers (with CRISPR) and then perform a large meiotic recombination mapping experiment than it would be to generate the lines required for CRISPR-mediated recombination mapping. It is possible to dramatically reduce costs by, for example, mapping sequentially at finer resolution. But this approach would require much more time than marker-assisted mapping. If someone develops a rapid and cheap method of reliably introducing DNA into Drosophila embryos, then this calculus might change.

However, it is possible to imagine situations where CRISPR-mediated mapping would be preferable, even for Drosophila. For example, some genomic regions display extremely low or highly non-uniform recombination rates. It is possible that CRISPR-mediated mapping could provide a reasonable approach to fine mapping genes in these regions.

The authors also propose the exciting possibility that CRISPR-mediated loss of heterozygosity could be used to map traits in sterile species hybrids. It is not entirely obvious to me how this experiment would proceed and I hope the authors can illuminate me. If we imagine driving a recombination event in the early embryo (with maternal Cas9 from one parent and gRNA from a second parent), then at best we would end up with chimeric individuals carrying mitotic clones. I don’t think one could generate diploid animals where all cells carried the same loss of heterozygosity event. Even if we could, this experiment would require construction of a substantial number of stable transgenic lines expressing gRNAs. Mapping an ~20Mbp chromosome arm to ~10kb would require on the order of two-thousand transgenic lines. Not an undertaking to be taken lightly. It is already possible to perform similar tests (hemizygosity tests) using D. melanogaster deficiency lines in crosses with D. simulans, so perhaps CRISPR-mediated LOH could complement these deficiency screens for fine mapping efforts. But, at the moment, it is not clear to me how to do the experiment.

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Translational Gene Editing – June 16-17, 2016 in Boston, MA

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Learn More | Sponsorship & Exhibit Details | Register by April 29 & SAVE up to $200!

IMPROVING CRISPR FOR BETTER FUNCTIONAL SCREENING

Optimized sgRNA Libraries for Genetic Screens with CRISPR-Cas9
John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MIT

Optimizing CRISPR for Pooled Genome-Wide Functional Genetic Screens
Paul Diehl, Ph.D., Director, Business Development, Cellecta, Inc.

CRISPR-Cas9 Whole Genome Screening: Going Where No Screen Has Gone Before
Ralph Garippa, Ph.D., Director, RNAi Core Facility, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center

Cross-Species Synthetic Lethal Screens and Applications to Drug Discovery
Norbert Perrimon, Ph.D., Professor, Department of Genetics, Harvard Medical School and Investigator, Howard Hughes Medical Institute

Interactive Breakout Discussion Groups with Continental Breakfast
This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants.

Topic: CRISPR/Cas9 System for In vivo Drug Discovery
Moderator: Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

  • Impact of CRISPR/Cas9 system on in vivo mouse models
  • Application of the CRISPR/Cas9 system in in vivo screens
  • Technical limitations/safety issues

Topic: Getting Past CRISPR Pain Points
Moderators: John Doench, Ph.D., Associate Director, Genetic Perturbation Platform, Broad Institute of Harvard and MITStephanie Mohr, Ph.D., Lecturer, Genetics & Director of the Drosophila RNAi Screening Center, Harvard Medical School

  • Challenges and solutions for CRISPR gRNA design
  • Methods for detecting engineered changes

Topic: Cellular Delivery of CRISPR/Cas9
Moderator: Daniel E Bauer M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

GENE EDITING FOR SCREENING DISEASE PATHWAYS AND DRUG TARGETS

Scouring the Non-Coding Genome by Saturating Edits
Daniel E. Bauer, M.D., Ph.D., Assistant Professor of Pediatrics, Harvard Medical School and Staff Physician in Pediatric Hematology/Oncology, Boston Children’s Hospital and Dana-Farber Cancer Institute, Principal Faculty, Harvard Stem Cell Institute

Parallel shRNA and CRISPR/Cas9 Screens Reveal Biology of Stress Pathways and Identify Novel Drug Targets
Michael Bassik, Ph.D., Assistant Professor, Department of Genetics, Stanford University

BUILDING THE CRISPR TOOLBOX

Beyond Cas9: Discovering Single Effector CRISPR Tools
Jonathan Gootenberg, Member, Laboratories of Dr. Aviv Regev and Dr. Feng Zhang, Department of Systems Biology, Harvard Medical School, and Broad Institute of Harvard and MIT

CRISPR-Cas9 Genome Editing Improves Sub-Cellular Localization Studies
Netanya Y. Spencer, M.D., Ph.D., Research Fellow in Medicine, Joslin Diabetes Center, Harvard Medical School

TECHNOLOGY PANEL: Trends in CRISPR Technologies
Panelists to be Announced

This panel will bring together 2-3 technical experts from leading technology and service companies to discuss trends and improvements in CRISPR libraries, reagents and platforms that users can expect to see in the near future. (Opportunities Available for Sponsoring Panelists)

APPLICATIONS OF CRISPR FOR DRUG DISCOVERY

Use of CRISPR and Other Genomic Technologies to Advance Drug Discovery
Namjin Chung, Ph.D., Head, Functional Genomics Platform, Discovery Research, AbbVie, Inc.

Application of Genome Editing Tools to Model Human Genetics Findings in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Genetics and Pharmacogenomics, Merck & Co. Inc.

In vivo Application of the CRISPR/Cas9 Technology for Translational Research
Danilo Maddalo, Ph.D., Lab Head, ONC Pharmacology, Novartis Institutes for BioMedical Research

DEVELOPING TOOLS FOR BETTER TRANSLATION

Improving CRISPR-Cas9 Precision through Tethered DNA-Binding Domains
Scot A. Wolfe, Ph.D., Associate Professor, Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School

Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing
Daniel G. Anderson, Ph.D., Professor, Department of Chemical Engineering, Institute for Medical Engineering & Science, Harvard-MIT Division of Health Sciences & Technology and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

Translating CRISPR/Cas9 into Novel Medicines
Alexandra Glucksmann, Ph.D., COO, Editas Medicine

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Curbing Cancer Cell Growth & Metastasis-on-a-Chip’ Models Cancer’s Spread

Curator: Larry H. Bernstein, MD, FCAP

 

New Approach to Curbing Cancer Cell Growth

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=189342

Using a new approach, scientists at The Scripps Research Institute (TSRI) and collaborating institutions have discovered a novel drug candidate that could be used to treat certain types of breast cancer, lung cancer and melanoma.

The new study focused on serine, one of the 20 amino acids (protein building blocks) found in nature. Many types of cancer require synthesis of serine to sustain rapid, constant and unregulated growth.

To find a drug candidate that interfered with this pathway, the team screened a large library of compounds from a variety of sources, searching for molecules that inhibited a specific enzyme known as 3-phosphoglycerate dehydrogenase (PHGDH), which is responsible for the first committed step in serine biosynthesis.

“In addition to discovering an inhibitor that targets cancer metabolism, we also now have a tool to help answer interesting questions about serine metabolism,” said Luke L. Lairson, assistant professor of chemistry at TSRI and principal investigator of cell biology at the California Institute for Biomedical Research (CALIBR).

Lairson was senior author of the study, published recently in the Proceedings of the National Academy of Sciences (PNAS), with Lewis Cantley of Weill Cornell Medical College and Costas Lyssiotis of the University of Michigan.

Addicted to Serine

Serine is necessary for nucleotide, protein and lipid biosynthesis in all cells. Cells use two main routes for acquiring serine: through import from the extracellular environment or through conversion of 3-phosphoglycerate (a glycolytic intermediate) by PHGDH.

“Since the late 1950s, it has been known that cancer cells use the process of aerobic glycolysis to generate metabolites needed for proliferative growth,” said Lairson.

This process can lead to an overproduction of serine. The genetic basis for this abundance had remained mysterious until recently, when it was demonstrated that some cancers acquire mutations that increased the expression of PHGDH; reducing PHGDH in these “serine-addicted” cancer cells also inhibited their growth.

The labs of Lewis C. Cantley at Weill Cornell Medical College (in work published in Nature Genetics) and David Sabatini at the Whitehead Institute (in work published in Nature) suggested PHGDH as a potential drug target for cancer types that overexpress the enzyme.

Lairson and colleagues hypothesized that a small molecule drug candidate that inhibited PHGDH could interfere with cancer metabolism and point the way to the development of an effective cancer therapeutic. Importantly, this drug candidate would be inactive against normal cells because they would be able to import enough serine to support ordinary growth.

As Easy as 1-2-800,000

Lairson, in collaboration with colleagues including Cantley, Lyssiotis, Edouard Mullarky of Weill Cornell and Harvard Medical School and Natasha Lucki of CALIBR, screened through a library of 800,000 small molecules using a high-throughput in vitro enzyme assay to detect inhibition of PHGDH. The group identified 408 candidates and further narrowed this list down based on cell-type specific anti-proliferative activity and by eliminating those inhibitors that broadly targeted other dehydrogenases.

With the successful identification of seven candidate inhibitors, the team sought to determine if these molecules could inhibit PHGDH in the complex cellular environment. To do so, the team used a mass spectrometry-based assay (test) to measure newly synthesized serine in a cell in the presence of the drug candidates.

One of the seven small molecules tested, named CBR-5884, was able to specifically inhibit serine synthesis by 30 percent, suggesting that the molecule specifically targeted PHGDH. The group went on to show that CBR-5884 was able to inhibit cell proliferation of breast cancer and melanoma cells lines that overexpress PHGDH.

As expected, CBR-5884 did not inhibit cancer cells that did not overexpress PHGDH, as they can import serine; however, when incubated in media lacking serine, the presence of CBR-5884 decreased growth in these cells.

The group anticipates much optimization work before this drug candidate can become an effective therapeutic. In pursuit of this goal, the researchers plan to take a medicinal chemistry approach to improve potency and metabolic stability.

 

How Cancer Stem Cells Thrive When Oxygen Is Scarce

(Image: Shutterstock)
image: Shutterstock

Working with human breast cancer cells and mice, scientists at The Johns Hopkins University say new experiments explain how certain cancer stem cells thrive in low oxygen conditions. Proliferation of such cells, which tend to resist chemotherapy and help tumors spread, are considered a major roadblock to successful cancer treatment.

The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say.

“There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells,” said Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center. “That gives us a few more possible targets for drugs that diminish their threat in human cancer.”

A summary of the findings was published online March 21 in the Proceedings of the National Academy of Sciences.

“Aggressive cancers contain regions where the cancer cells are starved for oxygen and die off, yet patients with these tumors generally have the worst outcome. Our new findings tell us that low oxygen conditions actually encourage certain cancer stem cells to multiply through the same mechanism used by embryonic stem cells.”

All stem cells are immature cells known for their ability to multiply indefinitely and give rise to progenitor cells that mature into specific cell types that populate the body’s tissues during embryonic development. They also replenish tissues throughout the life of an organism. But stem cells found in tumors use those same attributes and twist them to maintain and enhance the survival of cancers.

Recent studies showed that low oxygen conditions increase levels of a family of proteins known as HIFs, or hypoxia-inducible factors, that turn on hundreds of genes, including one called NANOG that instructs cells to become stem cells.

Studies of embryonic stem cells revealed that NANOG protein levels can be lowered by a chemical process known as methylation, which involves putting a methyl group chemical tag on a protein’s messenger RNA (mRNA) precursor. Semenza said methylation leads to the destruction of NANOG’s mRNA so that no protein is made, which in turn causes the embryonic stem cells to abandon their stem cell state and mature into different cell types.

Zeroing in on NANOG, the scientists found that low oxygen conditions increased NANOG’s mRNA levels through the action of HIF proteins, which turned on the gene for ALKBH5, which decreased the methylation and subsequent destruction of NANOG’s mRNA. When they prevented the cells from making ALKBH5, NANOG levels and the number of cancer stem cells decreased. When the researchers manipulated the cell’s genetics to increase levels of ALKBH5 without exposing them to low oxygen, they found this also decreased methylation of NANOG mRNA and increased the numbers of breast cancer stem cells.

Finally, using live mice, the scientists injected 1,000 triple-negative breast cancer cells into their mammary fat pads, where the mouse version of breast cancer forms. Unaltered cells created tumors in all seven mice injected with such cells, but when cells missing ALKBH5 were used, they caused tumors in only 43 percent (six out of 14) of mice. “That confirmed for us that ALKBH5 helps preserve cancer stem cells and their tumor-forming abilities,” Semenza said.

How cancer stem cells thrive when oxygen is scarce    https://www.sciencedaily.com/releases/2016/03/160328100159.htm

The new research, suggesting that low-oxygen conditions spur growth through the same chain of biochemical events in both embryonic stem cells and breast cancer stem cells, could offer a path through that roadblock, the investigators say.

“There are still many questions left to answer but we now know that oxygen poor environments, like those often found in advanced human breast cancers serve as nurseries for the birth of cancer stem cells,” says Gregg Semenza, M.D., Ph.D., the C. Michael Armstrong Professor of Medicine and a member of the Johns Hopkins Kimmel Cancer Center.

Chuanzhao Zhang, Debangshu Samanta, Haiquan Lu, John W. Bullen, Huimin Zhang, Ivan Chen, Xiaoshun He, Gregg L. Semenza.
Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA.
Proceedings of the National Academy of Sciences, 2016; 201602883     DOI: 10.1073/pnas.1602883113

Significance

Pluripotency factors, such as NANOG, play a critical role in the maintenance and specification of cancer stem cells, which are required for primary tumor formation and metastasis. In this study, we report that exposure of breast cancer cells to hypoxia (i.e., reduced O2 availability), which is a critical feature of the tumor microenvironment, induces N6-methyladenosine (m6A) demethylation and stabilization of NANOG mRNA, thereby promoting the breast cancer stem cell (BCSC) phenotype. We show that inhibiting the expression of AlkB homolog 5 (ALKBH5), which demethylates m6A, or the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α, which activate ALKBH5 gene transcription in hypoxic breast cancer cells, is an effective strategy to decrease NANOG expression and target BCSCs in vivo.

N6-methyladenosine (m6A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m6A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α–dependent expression of AlkB homolog 5 (ALKBH5), an m6A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m6A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.

Specific Proteins Found to Jump Start Spread of Cancer Cells

http://www.genengnews.com/gen-news-highlights/specific-proteins-found-to-jump-start-spread-of-cancer-cells/81252417/

Metastatic breast cancer cells. [National Cancer Institute]
http://www.genengnews.com/Media/images/GENHighlight/thumb_Feb29_2016_NCI_MetastaticBreastCancerCells1797514764.jpg

Scientists at the University of California, San Diego School of Medicine and Moores Cancer Center, with colleagues in Spain and Germany, have discovered how elevated levels of particular proteins in cancer cells trigger hyperactivity in other proteins, fueling the growth and spread of a variety of cancers. Their study (“Prognostic Impact of Modulators of G Proteins in Circulating Tumor Cells from Patients with Metastatic Colorectal Cancer”) is published in Scientific Reports.

Specifically, the international team, led by senior author Pradipta Ghosh, M.D., associate professor at the University of California San Diego School of Medicine, found that increased levels of expression of some members of a protein family called guanine nucleotide exchange factors (GEFs) triggered unsuspected hyperactivation of G proteins and subsequent progression or metastasis of cancer.

The discovery suggests GEFs offer a new and more precise indicator of disease state and prognosis. “We found that elevated expression of each GEF is associated with a shorter, progression-free survival in patients with metastatic colorectal cancer,” said Dr. Ghosh. “The GEFs fared better as prognostic markers than two well-known markers of cancer progression, and the clustering of all GEFs together improved the predictive accuracy of each individual family member.”

In recent years, circulating tumor cells (CTCs), which are shed from primary tumors into the bloodstream and act as seeds for new tumors taking root in other parts of the body, have become a prognostic and predictive biomarker. The presence of CTCs is used to monitor the efficacy of therapies and detect early signs of metastasis.

But counting CTCs in the bloodstream has limited utility, said Dr. Ghosh. “Enumeration alone does not capture the particular characteristics of CTCs that are actually tumorigenic and most likely to cause additional malignancies.”

Numerous efforts are underway to improve the value and precision of CTC analysis. According to Dr. Ghosh the new findings are a step in that direction. First, GEFs activate trimeric G proteins, and second, G protein signaling is involved in CTCs. G proteins are ubiquitous and essential molecular switches involved in transmitting external signals from stimuli into cells’ interiors. They have been a subject of heightened scientific interest for many years.

Dr. Ghosh and colleagues found that elevated expression of nonreceptor GEFs activates Gαi proteins, fueling CTCs and ultimately impacting the disease course and survival of cancer patients.

“Our work shows the prognostic impact of elevated expression of individual and clustered GEFs on survival and the benefit of transcriptome analysis of G protein regulatory proteins in cancer biology,” said Dr. Ghosh. “The next step will be to carry this technology into the clinic where it can be applied directly to deciphering a patient’s state of cancer and how best to treat.”

Metastasis-on-a-Chip’ Models Cancer’s Spread

http://www.mdtmag.com/news/2016/03/metastasis-chip-models-cancers-spread?et_cid=5200644&et_rid=461755519

In the journal Biotechnology Bioengineering, the team reports on its “metastasis-on-a-chip” system believed to be one of the first laboratory models of cancer spreading from one 3D tissue to another.

The current version of the system models a colorectal tumor spreading from the colon to the liver, the most common site of metastasis. Skardal said future versions could include additional organs, such as the lung and bone marrow, which are also potential sites of metastasis. The team also plans to model other types of cancer, such as the deadly brain tumor glioblastoma

To create the system, researchers encapsulated human intestine and colorectal cancer cells inside a biocompatible gel-like material to make a mini-organ. A mini-liver composed of human liver cells was made in the same way. These organoids were placed in a “chip” system made up of a set of micro-channels and chambers etched into the chip’s surface to mimic a simplified version of the body’s circulatory system. The tumor cells were tagged with fluorescent molecules so their activity could be viewed under a microscope.

To test whether the system could model metastasis, the researchers first used highly aggressive cancer cells in the colon organoid. Under the microscope, they saw the tumor grow in the colon organoid until the cells broke free, entered the circulatory system and then invaded the liver tissue, where another tumor formed and grew. When a less aggressive form of colon cancer was used in the system, the tumor did not metastasize, but continued to grow in the colon.

To test the system’s potential for screening drugs, the team introduced Marimastat, a drug used to inhibit metastasis in human patients, into the system and found that it significantly prevented the migration of metastatic cells over a 10-day period. Likewise, the team also tested 5-fluorouracil, a common colorectal cancer drug, which reduced the metabolic activity of the tumor cells.

“We are currently exploring whether other established anti-cancer drugs have the same effects in the system as they do in patients,” said Skardal. “If this link can be validated and expanded, we believe the system can be used to screen drug candidates for patients as a tool in personalized medicine. If we can create the same model systems, only with tumor cells from an actual patient, then we believe we can use this platform to determine the best therapy for any individual patient.”

The scientists are currently working to refine their system. They plan to use 3D printing to create organoids more similar in function to natural organs. And they aim to make the process of metastasis more realistic. When cancer spreads in the human body, the tumor cells must break through blood vessels to enter the blood steam and reach other organs. The scientists plan to add a barrier of endothelial cells, the cells that line blood vessels, to the model.

This concept of modeling the body’s processes on a miniature level is made possible because of advances in micro-tissue engineering and micro-fluidics technologies. It is similar to advances in the electronics industry made possible by miniaturizing electronics on a chip.

Scientists Synthesize Anti-Cancer Agent

A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University
A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University  http://www.dddmag.com/sites/dddmag.com/files/ddd1603_rice-anticancer.jpg

A team led by Rice University synthetic organic chemist K.C. Nicolaou has developed a new process for the synthesis of a series of potent anti-cancer agents originally found in bacteria.

The Nicolaou lab finds ways to replicate rare, naturally occurring compounds in larger amounts so they can be studied by biologists and clinicians as potential new medications. It also seeks to fine-tune the molecular structures of these compounds through analog design and synthesis to improve their disease-fighting properties and lessen their side effects.

Such is the case with their synthesis of trioxacarcins, reported this month in the Journal of the American Chemical Society.

“Not only does this synthesis render these valuable molecules readily available for biological investigation, but it also allows the previously unknown full structural elucidation of one of them,” Nicolaou said. “The newly developed synthetic technologies will allow us to construct variations for biological evaluation as part of a program to optimize their pharmacological profiles.”

At present, there are no drugs based on trioxacarcins, which damage DNA through a novel mechanism, Nicolaou said.

Trioxacarcins were discovered in the fermentation broth of the bacterial strain Streptomyces bottropensis. They disrupt the replication of cancer cells by binding and chemically modifying their genetic material.

“These molecules are endowed with powerful anti-tumor properties,” Nicolaou said. “They are not as potent as shishijimicin, which we also synthesized recently, but they are more powerful than taxol, the widely used anti-cancer drug. Our objective is to make it more powerful through fine-tuning its structure.”

He said his lab is working with a biotechnology partner to pair these cytotoxic compounds (called payloads) to cancer cell-targeting antibodies through chemical linkers. The process produces so-called antibody-drug conjugates as drugs to treat cancer patients. “It’s one of the latest frontiers in personalized targeting chemotherapies,” said Nicolaou, who earlier this year won the prestigious Wolf Prize in Chemistry.

Fluorescent Nanoparticle Tracks Cancer Treatment’s Effectiveness in Hours

Bevin Fletcher, Associate Editor    http://www.biosciencetechnology.com/news/2016/03/fluorescent-nanoparticle-tracks-cancer-treatments-effectiveness-hours

Using reporter nanoparticles loaded with either a chemotherapy or immunotherapy, researchers could distinguish between drug-sensitive and drug-resistant tumors in a pre-clinical model of prostate cancer. (Source: Brigham and Women's Hospital)

Using reporter nanoparticles loaded with either a chemotherapy or immunotherapy, researchers could distinguish between drug-sensitive and drug-resistant tumors in a pre-clinical model of prostate cancer. (Source: Brigham and Women’s Hospital)

Bioengineers at Brigham and Women’s Hospital have developed a new technique to help determine if chemotherapy is working in as few as eight hours after treatment. The new approach, which can also be used for monitoring the effectiveness of immunotherapy, has shown success in pre-clinical models.

The technology utilizes a nanoparticle, carrying anti-cancer drugs, that glows green when cancer cells begin dying. Researchers, using  the “reporter nanoparticles” that responds to a particular enzyme known as caspase, which is activated when cells die, were able to distinguish between a tumor that is drug-sensitive or drug-resistant much faster than conventional detection methods such as PET scans, CT and MRI.  The findings were published online March 28 in the Proceedings of the National Academy of Sciences.

“Using this approach, the cells light up the moment a cancer drug starts working,” co-corresponding author Shiladitya Sengupta, Ph.D., principal investigator in BWH’s Division of Bioengineering, said in a prepared statement.  “We can determine if a cancer therapy is effective within hours of treatment.  Our long-term goal is to find a way to monitor outcomes very early so that we don’t give a chemotherapy drug to patients who are not responding to it.”

Cancer killers send signal of success

Nanoparticles deliver drug, then give real-time feedback when tumor cells die   BY   SARAH SCHWARTZ

New lab-made nanoparticles deliver cancer drugs into tumors, then report their effects in real time by lighting up in response to proteins produced by dying cells. More light (right, green) indicates a tumor is responding to chemotherapy.

Tiny biochemical bundles carry chemotherapy drugs into tumors and light up when surrounding cancer cells start dying. Future iterations of these lab-made particles could allow doctors to monitor the effects of cancer treatment in real time, researchers report the week of March 28 in theProceedings of the National Academy of Sciences.

“This is the first system that allows you to read out whether your drug is working or not,” says study coauthor Shiladitya Sengupta, a bioengineer at Brigham and Women’s Hospital in Boston.

Each roughly 100-nanometer-wide particle consists of a drug and a fluorescent dye linked to a coiled molecular chain. Before the particles enter cells, the dye is tethered to a “quencher” molecule that prevents it from lighting up. When injected into the bloodstream of a mouse with cancer, the nanoparticles accumulate in tumor cells and release the drug, which activates a protein that tears a cancer cell apart. This cell-splitting protein not only kills the tumor cell, but also severs the link between the dye and the quencher, allowing the nanoparticles to glow under infrared light.

Reporter nanoparticle that monitors its anticancer efficacy in real time

Ashish Kulkarnia,b,1,Poornima Raoa,b,Siva Natarajana,b,Aaron Goldman, et al.
http://www.pnas.org/content/early/2016/03/28/1603455113.abstract

The ability to identify responders and nonresponders very early during chemotherapy by direct visualization of the activity of the anticancer treatment and to switch, if necessary, to a regimen that is effective can have a significant effect on the outcome as well as quality of life. Current approaches to quantify response rely on imaging techniques that fail to detect very early responses. In the case of immunotherapy, the early anatomical readout is often discordant with the biological response. This study describes a self-reporting nanomedicine that not only delivers chemotherapy or immunotherapy to the tumor but also reports back on its efficacy in real time, thereby identifying responders and nonresponders early on

The ability to monitor the efficacy of an anticancer treatment in real time can have a critical effect on the outcome. Currently, clinical readouts of efficacy rely on indirect or anatomic measurements, which occur over prolonged time scales postchemotherapy or postimmunotherapy and may not be concordant with the actual effect. Here we describe the biology-inspired engineering of a simple 2-in-1 reporter nanoparticle that not only delivers a cytotoxic or an immunotherapy payload to the tumor but also reports back on the efficacy in real time. The reporter nanoparticles are engineered from a novel two-staged stimuli-responsive polymeric material with an optimal ratio of an enzyme-cleavable drug or immunotherapy (effector elements) and a drug function-activatable reporter element. The spatiotemporally constrained delivery of the effector and the reporter elements in a single nanoparticle produces maximum signal enhancement due to the availability of the reporter element in the same cell as the drug, thereby effectively capturing the temporal apoptosis process. Using chemotherapy-sensitive and chemotherapy-resistant tumors in vivo, we show that the reporter nanoparticles can provide a real-time noninvasive readout of tumor response to chemotherapy. The reporter nanoparticle can also monitor the efficacy of immune checkpoint inhibition in melanoma. The self-reporting capability, for the first time to our knowledge, captures an anticancer nanoparticle in action in vivo.

 

Cancer Treatment’s New Direction  
Genetic testing helps oncologists target tumors and tailor treatments
http://www.wsj.com/articles/cancer-treatments-new-direction-1459193085

Evan Johnson had battled a cold for weeks, endured occasional nosebleeds and felt so fatigued he struggled to finish his workouts at the gym. But it was the unexplained bruises and chest pain that ultimately sent the then 23-year-old senior at the University of North Dakota to the Mayo Clinic. There a genetic test revealed a particularly aggressive form of acute myeloid leukemia. That was two years ago.

The harrowing roller-coaster that followed for Mr. Johnson and his family highlights new directions oncologists are taking with genetic testing to find and attack cancer. Tumors can evolve to resist treatments, and doctors are beginning to turn such setbacks into possible advantages by identifying new targets to attack as the tumors change.

His course involved a failed stem cell transplant, a half-dozen different drug regimens, four relapses and life-threatening side effects related to his treatment.

Nine months in, his leukemia had evolved to develop a surprising new mutation. The change meant the cancer escaped one treatment, but the new anomaly provided doctors with a fresh target, one susceptible to drugs approved for other cancers. Doctors adjusted Mr. Johnson’s treatment accordingly, knocked out the disease and paved the way for a second, more successful stem cell transplant. He has now been free of leukemia for a year.

Now patients with advanced cancer who are treated at major centers can expect to have their tumors sequenced, in hopes of finding a match in a growing medicine chest of drugs that precisely target mutations that drive cancer’s growth. When they work, such matches can have a dramatic effect on tumors. But these “precision medicines” aren’t cures. They are often foiled when tumors evolve, pushing doctors to take the next step to identify new mutations in hopes of attacking them with an effective treatment.

Dr. Kasi and his Mayo colleagues—Naseema Gangat, a hematologist, and Shahrukh Hashmi, a transplant specialist—are among the authors of an account of Mr. Johnson’s case published in January in the journal Leukemia Research Reports.

Before qualifying for a transplant, a patient’s blasts need to be under 5%.

To get under 5%, he started on a standard chemotherapy regimen and almost immediately, things went south. His blast cells plummeted, but “the chemo just wiped out my immune system,”

Then as mysteriously as it began, a serious mycotic throat infection stopped. But Mr. Johnson couldn’t tolerate the chemo, and his blast cells were on the rise. A two-drug combination that included the liver cancer drug Nexavar, which targets the FLT3 mutation, knocked back the blast cells. But the stem cell transplant in May, which came from one of his brothers, failed to take, and he relapsed after 67 days, around late July.

He was put into a clinical trial of an experimental AML drug being developed by Astellas Pharma of Japan. He started to regain weight. In November 2014, doctors spotted the initial signs in blood tests that Mr. Johnson’s cancer was evolving to acquire a new mutation. By late January, he relapsed again , but there was a Philadelphia chromosome mutation,  a well-known genetic alteration associated with chronic myeloid leukemia. It also is a target of the blockbuster cancer drug Gleevec and several other medicines.

Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3) inhibitor therapy with evolving actionable targets

Naseema GangatMark R. LitzowMrinal M. PatnaikShahrukh K. HashmiNaseema Gangat

Highlights
•   The article reports on a case of AML that underwent clonal evolution.
•   We report on novel acquisition of the Philadelphia t(9;22) translocation in AML.
•   Next generation sequencing maybe helpful in these refractory/relapse cases.
•   Novel FLT3-inhibitor targeted therapies are another option in patients with AML.
•   Personalizing cancer treatment based on evolving targets is a viable option.

For acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215) to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈https://clinicaltrials.gov/ct2/show/NCT02014558〉)

The development of kinase inhibitors for the treatment of leukemia has revolutionized the care of these patients. Since the introduction of imatinib for the treatment of chronic myeloid leukemia, multiple other tyrosine kinase inhibitors (TKIs) have become available[1]. Additionally, for acute myeloid leukemia (AML), identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3) has led to the development of several FLT3-inhibitors [2], [3], [4] and [5]. The article herein reports a unique case of AML that underwent clonal evolution while on a novel FLT3-inhibitor clinical trial.

Our work herein presents clinical and next generation sequencing data at the time of progression to illustrate these important concepts stemming from Darwinian evolution [6]. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process.

Our work focuses on a 23-year-old male who presented with 3 months history of fatigue and easy bruising, a white blood count of 22.0×109/L with 51% circulating blasts, hemoglobin 7.6 g/dL, and a platelet count of 43×109/L. A bone marrow biopsy confirmed a diagnosis of AML. Initial cytogenetic studies identified trisomy 8 in all the twenty metaphases examined. Mutational analysis revealed an internal tandem duplication of the FLT3 gene (FLT3-ITD).

He received standard induction chemotherapy (7+3) with cytarabine (ARA-C; 100 mg/m2for 7 days) and daunorubicin (DNM; 60 mg/m2 for 3 days). His induction chemotherapy was complicated by severe palatine and uvular necrosis of indeterminate etiology (possible mucormycosis).

Bone marrow biopsy at day 28 demonstrated persistent disease with 10% bone marrow blasts (Fig. 1). Due to his complicated clinical course and the presence of a FLT3-ITD, salvage therapy with 5-azacitidine (5-AZA) and sorafenib (SFN) was instituted. Table 1.
The highlighted therapies were employed in this particular case at various time points as shown in Fig. 1.

http://ars.els-cdn.com/content/image/1-s2.0-S221304891530025X-gr1.jpg

References

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    • Nature, 481 (7381) (2012), pp. 306–313

 

 

 

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