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Live Conference Coverage AACR 2020 in Real Time: Monday June 22, 2020 Mid Day Sessions

Reporter: Stephen J. Williams, PhD

This post will be UPDATED during the next two days with notes from recordings from other talks

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AACR VIRTUAL ANNUAL MEETING II

 

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.

 

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Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Research

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.

John E. Dick, Enzo Galligioni, David A Tuveson

DETAILS

Awardee: John E. Dick
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

Chemistry to the Clinic: Part 1: Lead Optimization Case Studies in Cancer Drug Discovery

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

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Educational Session

Bioinformatics and Systems Biology, Molecular and Cellular Biology/Genetics

Informatics Technologies for Cancer Research

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

DETAILS

  • Variant analysis is the big bottleneck, especially interpretation of variants
  • CIVIC resource is a network for curation, interpretation of genetic variants
  • CIVIC curators go through multiple rounds of editors review
  • gene summaries, variant summaries
  • curation follows ACSME guidelines
  • evidences are accumulated, categories by various ontologies and is the heart of the reports
  • as this is a network of curators the knowledgebase expands
  • CIVIC is linked to multiple external informatic, clinical, and genetic databases
  • they have curated 7017 clinical interpretations, 2527 variants, using 2578 papers, and over 1000 curators
  • they are currently integrating with COSMIC ClinVar, and UniProt
  • they are partnering with ClinGen to expand network of curators and their curation effort
  • CIVIC uses a Python interface; available on website

https://civicdb.org/home

The Precision Medicine Revolution

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

Towards FAIR data in cancer imaging research

Andriy Fedorov, PhD

Towards the FAIR principles

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

Engineering and Physical Sciences Approaches in Cancer Research, Diagnosis, and Therapy

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

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Educational Session

Survivorship

Exceptional Responders and Long-Term Survivors

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

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Educational Session

Tumor Biology, Immunology

Exploiting Metabolic Vulnerabilities in Cancer

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

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Educational Session

Other Articles on this Open Access  Online Journal on Cancer Conferences and Conference Coverage in Real Time Include

Press Coverage

Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Symposium: New Drugs on the Horizon Part 3 12:30-1:25 PM

Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Session on NCI Activities: COVID-19 and Cancer Research 5:20 PM

Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Session on Evaluating Cancer Genomics from Normal Tissues Through Metastatic Disease 3:50 PM

Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Session on Novel Targets and Therapies 2:35 PM

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Reengineering Therapeutics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

The synNotch solution: UCSF scientists engineer a next-gen T-cell immunotherapy

Sunday, January 31, 2016 | By John Carroll

2.2.9

2.2.9   Reengineering Therapeutics, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

CAR-T has been all the rage in cancer R&D for several years now as a slate of biotech upstarts pursue highly promising work reengineering T cells into attack weapons by adding a chimeric antigen receptor that can zero in on particular cancer cells. The approach has been highly effective in acute lymphoblastic leukemia, triggering an attack on B cells by homing in on the CD19 antigen, a breakthrough that has inspired a race to the regulatory finish line with the first CAR-Ts.

That approach, though, has run into some major obstacles when researchers move from the blood cancer to solid tumors. But now a group of scientists working with UC San Francisco’s Wendell Lim say they’ve come up with a new therapeutic model for T-cell engineering that promises to overcome that hurdle and make it a more precise weapon that can tackle solid tumors while avoiding off-target reactions that threaten patients.

The key to this new approach is a new receptor: synNotch. Taking a cue from nature, which relies on a sensor called Notch to perform key functions, the synthetic biology engineers say they can add a receptor that includes one section that sticks out from the cell with one that lies inside. By tinkering with synNotch they can reengineer the immune cell to run down a particular cancer cell target and then issue instructions to turn genes on or off to set up the other half of the therapeutic equation.

In a project described in Cell, the team says they created a synNotch that recognized an antigen on the surface of the cancer cell while the internal mechanism contributed a chimeric antigen receptor that recognized a different antigen. They then tested it on a mouse model that included two different tumor cells: one with both targets recognized by the external synNotch sensor and the CAR and one with just the CAR target.

The newly programmed attack weapon zeroed in on the two targets (which required synNotch activation for it to work) while leaving the other alone, providing preclinical proof-of-concept evidence that they could create a much more efficient tumor cell killing vehicle.

“The kinds of engineered T cells that we can now construct give us the exciting potential to create precision cancer therapeutics that take advantage of all the genomic and proteomic information we are currently gathering on disease,” said Lim, the senior author of the study. “This genomic information now becomes actionable.”

The team was led by Leonardo Morsut and Kole Roybal.

Lim added that it is possible to reengineer a T cell with multiple synNotches to make it even more precise. And there are added applications for autoimmune disease, regenerative medicine, and more.

Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

Leonardo  MorsutKole T. Roybal, Xin Xiong, Russell M. Gordley, Scott M. Coyle, Matthew Thomson, Wendell A. Lim

Tricked-Out Immune Cells Could Attack Cancer, Spare Healthy Cells  

New Cell-Engineering Technique May Lead To Precision Immunotherapies

New CRISPR/Cas9 delivery method could offer a clinical pathway

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Enhanced Cas9 for more precise editing

Larry H. Bernstein, MD, FCAP, Curator

LPBI

2.2.13

2.2.13   Enhanced Cas9 for more precise editing, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

CRISPR-Cas9 Genome Editing Hurdle Overcome

http://www.technologynetworks.com/Genomics/news.aspx?ID=185830

Team re-engineers system to dramatically cut down on editing errors; improvements advance future human applications.

MIT-Crispr_0.jpg

http://www.technologynetworks.com/images/videos/News%20Images/GR/MIT-Crispr_0.jpg

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on “off-target” editing errors. The refined technique addresses one of the major technical issues in the use of genome editing.

The CRISPR-Cas9 system works by making a precisely targeted modification in a cell’s DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, it can bind to and cut additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a gene out entirely, which might lead to the development of cancer or other problems.

advance future human applications.
Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on “off-target” editing errors. The refined technique addresses one of the major technical issues in the use of genome editing.

The CRISPR-Cas9 system works by making a precisely targeted modification in a cell’s DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, it can bind to and cut additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a gene out entirely, which might lead to the development of cancer or other problems.

MIT-Crispr_0.jpg

In a recently published paper Feng Zhang and his colleagues report that changing three of the approximately 1,400 amino acids that make up the Cas9 enzyme from S. pyogenes dramatically reduced “off-target editing” to undetectable levels in the specific cases examined. Zhang is the W.M. Keck Career Development Professor in Biomedical Engineering in MIT’s departments of Brain and Cognitive Sciences and Biological Engineering, and a member of both the Broad Institute and McGovern Institute.

Zhang and his colleagues used knowledge about the structure of the Cas9 protein to decrease off-target cutting. DNA, which is negatively charged, binds to a groove in the Cas9 protein that is positively charged. Knowing the structure, the scientists were able to predict that replacing some of the positively charged amino acids with neutral ones would decrease the binding of “off target” sequences much more than “on target” sequences.

After experimenting with various possible changes, Zhang’s team found that mutations in three amino acids dramatically reduced “off-target” cuts. For the guide RNAs tested, “off-target” cutting was so low as to be undetectable.

The newly-engineered enzyme, which the team calls “enhanced” S. pyogenes Cas9, or eSpCas9, will be useful for genome editing applications that require a high level of specificity. The Zhang Lab is immediately making the eSpCas9 enzyme available for researchers worldwide. The team believes the same charge-changing approach will work with other recently described RNA-guided DNA targeting enzymes, including Cpf1, C2C1, and C2C3, which Zhang and his collaborators reported on earlier this year.

The prospect of rapid and efficient genome editing raises many ethical and societal concerns, says Zhang, who is speaking this morning at the International Summit on Gene Editing in Washington. “Many of the safety concerns are related to off-target effects,” he says. “We hope the development of eSpCas9 will help address some of those concerns, but we certainly don’t see this as a magic bullet. The field is advancing at a rapid pace, and there is still a lot to learn before we can consider applying this technology for clinical use.

Zhang Lab Produces Engineered CRISPR Complex with Greater Precision

Aaron Krol

http://www.bio-itworld.com/2015/12/3/zhang-lab-produces-engineered-crispr-complex-greater-precision.html

CRISPR-Cas9 gene editing has quickly become a basic tool of molecular biology, but don’t let that fool you into thinking we know exactly how it works. Armed with the DNA-slicing Cas9 molecule and a single guide RNA (sgRNA) sequence, biologists can and enthusiastically do make small, precise cuts almost anywhere in the genome of any species, altering the genetic code at will. But the steps to the intricate molecular tango of Cas9 and DNA ― and the reasons their footwork sometimes slips ― are still being worked out.

In fact, while Jennifer Doudna and Emmanuelle Charpentier first demonstrated how to program Cas9 for gene editing in 2012, it wasn’t until early last year that they were able to propose a 3D structure for the molecule, based on x-ray crystallography images. Knowing the shape of Cas9, and which substructures it uses to link hands with DNA, is crucial to learning how it makes its cuts. (Interestingly, at least one group, working at Montana State University, had gotten images of a CRISPR-associated molecule as early as 2010.)

As a new report published this week in Science shows, Cas9 will only become more powerful as we puzzle out exactly how it interacts with DNA. The paper, a product of Feng Zhang’s lab at the Broad Institute of MIT and Harvard, presents a tweaked version of Cas9 that slightly loosens its grip on DNA. It’s a feat of “rational engineering,” using knowledge of a molecule’s form and function to deliberately change its behavior ― as opposed to the random plug-and-play search for better molecules that has sometimes characterized CRISPR-Cas9 research.

Zhang and his colleagues, including lead author Ian Slaymaker, offer their new Cas9 as a solution to the problem of off-target cuts, where an sgRNA sequence mistakenly sets up the Cas9 guillotine over the wrong area of the genome. These misdirected cuts crop up at low levels in almost every CRISPR-Cas9 experiment. In the lab they’re basically harmless, and most fall across meaningless strings of DNA that the genome can easily do without. But if we were to start using CRISPR as a therapy, to correct rare diseases embedded in people’s DNA, the one-in-a-hundred chance of snipping out something vital becomes an all too real threat.

Zhang has a big stake in wiping out off-target errors. Not only is his lab a powerhouse of CRISPR research (he’s sometimes mentioned as a likely contender for a Nobel Prize in connection with the technology), he is also a scientific founder of Editas Medicine, one of three companies hoping to get Cas9-based cures into the clinic. So it’s not surprising to see his group scouring the structure of Cas9 for ways to make it more discerning about the DNA it chops up.

Holding On With Both Hands

The Science paper started with an observation about how Cas9 contends with damaged DNA.

The authors noticed that, when the target DNA itself is weakened by a mismatch between its two strands, even poorly designed Cas9 complexes are able to cut it effectively. It seemed that a gap between DNA strands provided an opening for a Cas9 molecule that wouldn’t otherwise be up to the job.

They reasoned that Cas9 needs to separate both DNA strands before it can slice them ― and that a Cas9 molecule that strains to separate those strands would be less promiscuous with its scissors.

Cas9 appears to attach to DNA at two junctions on opposite strands. First, Cas9 uses an sgRNA molecule to bind to a specified sequence of DNA; that’s the reason its edits can be aimed so precisely. Close but imperfect sgRNA matches are what cause off-target cuts: a mismatch will weaken Cas9’s grip on the DNA molecule, but not always enough to stop it from pulling the two strands apart, especially if it’s found a part of the genome with many similarities to its real target.

Second, once Cas9 has latched on to its DNA target, it pulls at the opposite strand by clutching it in a groove between two structures called the RuvC and HNH domains. This second grip doesn’t care about the DNA sequence at all. Instead, the groove has a strong positive charge, which creates a powerful bond with the negatively charged DNA.

http://www.bio-itworld.com/uploadedImages/Bio-IT_World/Top_Headlines/2015/12-Dec/Cas9%20Image.jpg

Images of the Cas9 molecule, with RuvC and HNH domains colored, show how it interacts with sgRNA and DNA to pull DNA strands apart before cutting them. Image credit: Broad Institute

That was the inspiration for the Zhang lab’s engineering of a new Cas9 molecule. The team found 32 sites inside the RuvC-HNH groove where they could replace positively-charged components with alanine, a neutrally-charged amino acid. One by one, they made these switches to create 32 different mutant Cas9 molecules, each of which should theoretically have had a weaker clutch than the natural version. The lab reasoned that this would make a perfect sgRNA match even more important. In an off-target match, already weakened by a poor bond between DNA and sgRNA, the two DNA strands might be too hard to separate for Cas9 to make its cut.

The hunch was borne out when the Zhang lab used its mutant Cas9 molecules to make cuts that they knew would normally cause several off-target glitches, and checked whether their new molecules were making the same mistakes. After a few rounds of experiments, they started mixing the most promising mutations together, searching for a Cas9 molecule that performed well in multiple gene regions known to be problematic for natural Cas9.

Finally, the authors settled on a modified Cas9 molecule they named “eSpCas9,” which appears to cause especially few off-target cuts, without dulling its blade for its real DNA targets. This new molecule actually has three different alanine substitutions, a product of both rational engineering and simple trial-and-error.

Inching Toward the Clinic

The Broad Institute will be releasing eSpCas9 for outside scientists to use, something it has also done with past CRISPR innovations like libraries of useful sgRNA sequences. Future studies, however, covering more tricky regions across the genome, will probably turn up even more precise Cas9 variants; we may even find that different Cas9 mutants are better suited for different tasks.

Zhang himself has been very interested in the search for new and better Cas9 molecules for particular jobs. Just this April, his lab published research on the incredible variety of CRISPR complexes found in nature, where hundreds of bacterial species use their own molecular armaments to slice up the DNA of invading viruses. While most work with Cas9 has used a molecule found in Streptococcus pyogenes, Zhang and Editas have high hopes for Staphylococcus aureus Cas9 ― a version of the molecule so compact that it can be encoded in a virus, the better to deliver to patients’ cells for Cas9-based therapies.

Indeed, while the experiments described in the Science paper all work with S. pyogenes Cas9, the authors also note that they’ve performed similar fine-tuning of Cas9 molecules from other bacteria, S. aureus among them. So while scientists around the world may find eSpCas9 very useful in the lab, it’s a fair guess that, at Editas, the research staff will be following other avenues from this research.

Meanwhile, Zhang and several of his co-authors are currently in Washington, D.C., at a summit to discuss the future of gene editing in light of CRISPR-Cas9’s fast-evolving possibilities. For now, most experts would agree that risks from off-target errors make gene editing of humans a dangerous proposition. But these problems are swiftly clearing up ― leaving the ethical and political questions around engineering the human genome more salient than ever before.

Enhanced Cas9 Enzyme Keeps Gene Editing On Target

  • Structure-guided mutagenesis has guided the engineering of an “enhanced specificity”Streptococcus pyogenes Cas9. In the newly engineered Cas9, three positively charged amino acids from the enzyme’s positively charged, DNA-cradling groove have been replaced by neutral amino acids. With the modified Cas9, the binding of on-target sites appears to be weakened much less than the binding of off-target sites. [Ian Slaymaker, Broad Institute]

    The CRISPR-Cas9 gene-editing system has been known to be overzealous, unwilling to call it quits after cutting its target site. Unfortunately, in many cases, CRISPR-Cas9 won’t leave well enough alone. It persists inside a cell, cutting additional sites, introducing unwanted edits, and propagating “off target” effects.

    A calmer CRISPR-Cas9, reasoned scientists at the Broad Institute, might make a finer editing tool, one less given to coloring outside the lines. These scientists, led by Feng Zhang, Ph.D., started tinkering with the CRISPR-Cas9’s business end, the DNA-snipping Cas9 endonuclease. Ultimately, they developed a Cas9 that binds more weakly, whether its binding partner is an on-target or off-target stretch of DNA. Yet they were able to ensure that the binding of on-target sites was weakened much less than the binding of off-target sites.

    The approach used by Dr. Zhang and colleagues involved swapping out some of Cas9’s 1,400-or-so amino acids. Cas9 has positively charged amino acids that form a positively charged groove that cradles positively charged stretches of DNA. By removing some of these positively charged amino acids and replacing them with neutral amino acids, the scientists hoped to create a less grabby, cut-happy Cas9.

Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity.

Science (2015)   http://dx.doi.org:/10.1126/science.aad5227  (Epub ahead of print).

CRISPR Reduces Cancer Genomes to Bare Essentials

http://www.genengnews.com/gen-news-highlights/crispr-reduces-cancer-genomes-to-bare-essentials/81252034/

Crystal structure of Cas9 in complex with guide RNA and target DNA. [Hiroshi Nishimasu et al./Wikipedia]
http://www.genengnews.com/Media/images/GENHighlight/thumb_Nov30_2015_Wikipedia_CrystalStructureCas92302235168.jpg

To identify weaknesses in cancer that can be targeted with new therapies, several teams of scientists are working to cut cancer genomes down to size, gene by gene. The latest team to wield the genomic broadsword is based at the University of Toronto. This team, led by Jason Moffat, Ph.D., has used a technique called deletion mutant analysis to switch off, one by one, almost 18,000 genes—almost 90% of the entire human genome. This winnowing process, which relies on the CRISPR gene-editing technique and is called deletion mutant analysis, allowed the team to identify a core set of 1,580 human core fitness genes. Moreover, the team whittled away at the genomes of five different cancer cell lines to find context-dependent fitness genes.

“It’s when you get outside the core set of essential genes, that it starts to get interesting in terms of how to target particular genes in different cancers and other disease states,” explained Dr. Moffat.

The new work appeared November 25 in the journal Cell, in an article entitled “High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.” It follows similar studies that appeared online last month in the journal Science. Both of these studies, which appeared in print November 27, reported a consistent set of about 2,000 genes that are indispensable for viability in human cells. (For a news report about one of these studies, see “CRISPR-Fine Screen of Human Genome Identifies Essential Genes.”)

The new findings in Cell show that:

  • Core fitness genes are highly enriched for ancient protein complexes.
  • Context-specific fitness genes illuminate biological differences between cell types.
  • Distinct genetic signatures can be used to predict differential drug response.

Moreover, the new findings show the majority of human genes play more subtle roles in the cell because switching them off doesn’t kill the cell. But if two or more of such genes are mutated at the same time, or the cells are under environmental stress, their loss begins to count.

Because different cancers have different mutations, they tend to rely on different sets of genes to survive. By turning genes off in five different cancer cell lines, including brain, retinal, ovarian, and two kinds of colorectal cancer cells, Dr. Moffatt’s team learned that each tumor relies on a unique set of genes that can be targeted by specific drugs. Essentially, the team identified distinct sets of “smoking gun” genes for each of the tested cancers. Each set may prove to be susceptible to different drugs.

“[We] demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds,” wrote the authors of the Cell article. “Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.”

The authors also demonstrated that distinct genetic signatures can be used to predict differential drug response. Specifically, they found that metformin, a widely prescribed diabetes drug, successfully killed brain cancer cells and those of one form of colorectal cancer. The same drug, however, was useless against the other cancers studied.

Similarly, the antibiotics chloramphenicol and linezolid were effective against one form of colorectal cancer, but not against brain or other cancers studied. These data illustrate the clinical potential of the data in pointing to more precise treatments for the different cancers—and suggest the value of personalized medicine.

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