Posts Tagged ‘cancer’

Second Phase SBIR Contract from the National Cancer Institute (NCI) to Identify Lethal Gene Combinations in Cancer Cell Models

October 23rd, 2012
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It’s been a while since the last post to this blog. It is not that nothing has been going on, in fact, quite the opposite. It has been quite busy the past several months and, unfortunately, blog postings have suffered. However, I thought I would get things started again with short post about our recent SBIR Contract.

Cellecta has been awarded an SBIR Phase II Contract from the NIH National Cancer Institute to continue its work using RNAi screens to identify paired combinations of DNA damage and repair (DDR) genes essential for cancer cells. Some results from the first phase of the grant were posted previously: http://cellecta.com/blog/2011/08/31/rnai-screen-cancer-synergistic-lethality. That work was essentially a proof of principle targeting combinations of 40 DDR genes. With this continuation of the contract, we can now move into a full scale screen of over 400 different DDR genes and run the screen in multiple cell models so we can identify which gene combinations are essential in each.

The purpose of the screen is to identify synthetic lethal genes–genes that, when inactivated together, have a significantly stronger lethal effect than when either is inactivated alone. The idea is that combination therapies could be developed using drugs that target both of the genes together. This combination approach makes is less likely for cancer cells to develop resistance to treatment.

With array based screening, however, it is extremely resource and labor intensive to identify these lethal combinations. However, a pooled RNAi screening approach using a library that co-expresses pairs of shRNAs targeting all combinations of the DDR genes enables efficient screens of tens of thousands of combinations.

We are making the dual shRNA expression libraries now and looking forward to getting these combinational screens started as soon as possible. As soon as we have something interesting, I’ll post it here—so keep an eye out for updates.
You can read the press release about Cellecta’s new SBIR Contract at http://www.prweb.com/releases/20121023/cellecta-rnai-screening/prweb10043967.htm

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Posted in Cancer, NIH, Pooled shRNA Library, RNAi Screening, Synergistic Lethality, Synthetic Lethality

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The Need for RNAi Screening Standards

February 3rd, 2012
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A couple of months ago at the CHI Discovery on Target Conference, Hakim Djaballah, Director of the HTS Core Facility at the Memorial Sloan Kettering Cancer Center, gave a unique and insightful presentation highlighting the challenges RNAi screening to identify lethal loss of function interaction in oncogenic systems.

For the exceptional benefits RNAi offers as a targeted tool to elucidate gene function, it is still a relatively new technology with limitations, potential, and idiosyncrasies that remain somewhat undefined.  These features are especially evident when RNAi is adapted for large-scale screening of gene function where small details in the set-up, screening process, quality of the reagents, and types of cells can significantly affect the variation and consistency in the large amount of data generated.  By highlighting some disappointing follow-up results from initially exciting high-profile publications, Dr. Djaballah identified a few critical benchmarks for evaluating RNAi screening.

Dr. Djaballah’s group looked at three potentially high value cancer targets identified in independent loss-of-function RNAi screens.  In addition to the publications, his group reviewed the primary screening data in more detail to evaluate the procedure and statistical significance of the data. The first screen, published in May 2009 in Cell (Scholl, et al.), identified STK33 as required for KRAS oncogenic activity.  Initially, a very exciting discovery, a number of groups pursued STK33 as a potential therapeutic target.  However, subsequent groups (Barbie, et al. and Luo, et al.) failed to find the STK33 among the strong hits in similar screens.  A recent publication in September 2011 by Babij, et al. in Cancer Research, which also does not see STK33 as a hit in a similar shRNA screen, presents compelling data indicating STK33 is not, in fact, generally essential for survival KRAS-dependant cells.  Although, based on recent letters to the editor in Cancer Research, this does not seem to be the end of the discussion between these two groups, the initial excitement of STK33 seems to have been premature at best.

The KRAS synthetic lethal screens by Barbie et al. mentioned above that did not pick-up STK33 as a strong hit their screen did, however,  identify another potentially interesting gene—the IκB kinase TBK1—that appeared essential for, but previously unknown to be involved in,  KRAS lethality.  This target was not found by other groups and has yet to be confirmed, but Dr. Djaballah had some reservations as to whether statistical analysis of the data really supported this as a true “hit” or simply an outlier.  Dr. Djaballah also had similar concerns with a recent Nature Letter in Oct. 2011 by Zuber et al that identified Brd4 as an essential gene and possible therapeutic target in acute myeloid leukemia cells.  A more detailed review of the data from this screen revealed some issues with the confidence of this hit, as there was significant variability in the CV values and a couple of the shRNAs were enriched by as much as million fold.

Dr. Djaballah’s discussion was clearly not intended to disparage any specific study, but rather to demonstrate the slippery potential of over-interpreting the extensive data produced by such a powerful approach.  Based on the amount of resources and effort put into a complex screen, it can be difficult to maintain the reserve required to coldly and rigorously analyze the experimental design and results in a detached manner and properly assess which candidates really meet the criteria for follow up.  As a relatively new screening technology without much in the way of standards and defined good practices, it is easy to prematurely “fall in love” with potentially interesting targets that may be just noise in the data.  From his analysis, Dr. Djaballah suggests paying particular attention to the following three aspects:

  1. Do the infections at the appropriate MOI and with sufficient cells to assess the effect. Although Dr. Djaballah was primarily talking about arrayed screens (with single shRNA plasmids in wells), the points is also very valid for pooled screening.  It is critical to ensure there are each cells containing each shRNA to be assayed to generate reliable reproducible results.
  2. Use correct passage times. Whether the screen requires looking at knockdowns or survival, it is important that the cells are maintained for a long enough passage number to produce a significant differential between affected and non-affected cells.  Conversely, too many passages will introduce too much noise.
  3. Pay attention to general data and overall results. It is important to see if the overall screening results make sense.  For example, are known lethal genes showing up in the hits?

While pointing out the importance of these considerations in evaluating a screening, Dr. Djaballah contended overall that there are currently few standards of practice to provide guidelines around these and similar procedural details.  We at Cellecta would agree, having focused most of our effort in the last several years toward optimizing the many subtleties of pooled shRNA screening to enable consistent, robust, and interpretable results.

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Posted in Article, Cancer, Conferences, Pooled shRNA Library, RNAi Screening, Technical

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Tissue Targeting May Offer an Alternative Therapeutic Approach for Difficult-to-Treat Diseases

October 13th, 2011
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We recently received a phase II of our SBIR grant Exploiting Synthetic Lethality of Hematopoietic Lineage Cells to Develop Novel Targets from the NIH. Rather than trying to identify potential drug targets in oncogenic hematopoietic cells, much of the effort for this project focuses on trying to develop a pharmacologic approach to identify and kill off all hematopoietic cells (see recent press release). This sort of capability may offer an alternative therapeutic approach relying on tissue ablation and renewal to treat hematopoietic cancers such as leukemia and lymphoma.

Clinical approaches exist to regrow and regenerate portions of many essential tissues. For serious diseases, this capability offers a somewhat aggressive treatment possibility where affected tissues are completely eliminated and replaced by new healthy tissue. Blood is one such tissue where that can be regenerated with current clinical procedures. Though risky, a patient’s blood can be regenerated from a bone marrow cell graft through autologous hematopoietic stem cell transplantation and this is a currently a treatment of last resort for individuals suffering with life-threatening blood or bone marrow cancers. However, there is also much focus on regenerative approaches with other tissues, such as bone and skin. In addition, loss of other tissues such as thymus, prostate, and ovary do not have a significant negative impact on the quality of life. As research advances, it is reasonable to assume regenerative approaches will be available for an increasing range of cell types and tissues.

Since all cells of one tissue or lineage type are removed and replaced using this approach, the specific pathology is not particularly significant for ablative and regenerative treatments. Rather than targeting specific cells based on certain disease biology, eradication of all cells in a particular class eliminates the disorder regardless of its nature. This opens up a real opportunity to develop effective therapies for a range of diseases for which there are currently limited treatment options. As clinical technology develops, stem cell therapies improves, more tissue regeneration protocols are established, and in vitro tissue and organ culture technology becomes routine, ablative/replacement treatments may become the preferred therapeutic approach to treat any number of a broad range of disease states.

A major hurdle with this sort of therapy, however, is the in situ eradication step of the diseased cell or tissue. Currently, the principal ways to eliminate damaged cells types or tissues are through localized excision via surgery or radioactive ablation. For example, with autologous hematopoietic stem cell transplantation mentioned above, much of the toxicity of the treatment is associated with the general full-body radiation treatments to which patients are subjected to ablate an individual’s endogenous bone marrow before grafting in new healthy tissue. More precisely targeted approaches to eliminate affected cells are necessary if tissue replacement is to become a generally useful treatment option. Pharmacologicals that target and kill specific types of cells would provide a much needed solution for this problem, and may be easier to develop than drugs that specifically target only diseased, but not healthy, cells. The first step in developing these sorts of targeted molecules is identifying unique tissue-specific markers and potential drug targets as we are attempting for hematopoietic cells with this project.

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Posted in Cancer, NIH, Pooled shRNA Library, RNAi Screening, Synergistic Lethality, Synthetic Lethality

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Screening for Synergistically Lethal Knockdown Combinations in Cancer Cells

August 31st, 2011
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Therapeutic approaches using multiple drug combinations have become a standard treatment model for many types of cancer. Due to the tremendous genetic complexity and adaptive nature of most human malignancies, the use of multiple drugs acting on different targets increases the efficacy and helps thwart the development of drug resistance. However, the search for new treatment options and expanding number of drug candidates create a demand for better understanding and prediction of the most effective combinations to expedite evaluation and application in clinical settings.

To help address this challenge, Cellecta responded to an NIH contract request to develop new tools that help assess the effect of combinatorially silencing pairs of genes. We developed a variation of our RNAi pooled screening that systematically identifies and prioritizes gene pairs that, when knocked down, significantly inhibit cancer cell growth. In other words, rather than simply identifying which individual genes are essential for growth of cancer cell lines, we identify which pairs that, when silenced, most significantly inhibit cancer cell proliferation.

In some cases, the loss of two genes may be additive and strongly impair cell growth much more significantly than the loss of either gene independently. In fact, sometimes either gene independently may not have any negative effect on cells but, when both are knocked down, there is a synergistic effect that is very lethal to cells. Conversely, losing the function of two known essential genes may not, in fact, have any more of an adverse affect on cell proliferation than the loss either separately. As a result, then, it is very difficult to predict the effect of a loss of a pair of genes so each combination must be tested.

For this project, we made a specialized lentiviral vector containing two shRNA expression cassettes so the construct expresses two different shRNAs from independent promoters. A library of shRNAs was cloned into each of these shRNA expression cassettes to make a pooled heterogeneous population that expressed all paired combinations of shRNAs. With some cloning tricks, we were able to incorporate a short uniquely identifiable sequence (i.e., a “bar-code”) that identified which two shRNAs were in each vector.

The data below were generated with four shRNAs designed against each of 40 DNA damage and repair genes (160 shRNAs total) so, on completion, there were 25,600 different combinations—160 in the first shRNA position vs. 160 in the second. Using this library, we ran an RNAi lethality screen with an isogenic panel of immortalized human mammary epithelial (HMEC) cells using our standard procedures. We have validated several of the pairs and confirmed the combinatorial effect on cell growth. The approach can be reasonably extended to systematically test all combinations of approximately 200 targets in a single screen.

 
Cytotoxicity level of shRNA combinations identified in synergistic lethality screen with 27K DDR library in HMEC-TERT cells.
 

Cytotoxicity (bar-code depletion) level of bispecific shRNA constructs identified in SL screen with 27K DDR library in HMEC-TERT cells. Control – shRNA targeting luciferase gene.
 

Obviously, this approach provides an alternative to what would otherwise be extremely time consuming and expensive pair-wise individual assays to assess lethal gene knockdown combinations in large numbers of target genes. Moreover, it demonstrates the power and flexibility of pooled library screens to address the challenges of elucidating the multiple functional roles and importance of various genes in the variety of biological model systems used in life science and drug discovery research.

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Posted in Cancer, NIH, Pooled shRNA Library, RNAi Screening, Synergistic Lethality

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AACR-NCI Systems Biology Conference

March 11th, 2011
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Alex Chenchik, Cellecta’s founder and Scientific Director, and Paul Diehl, Director of Business Development, attended the AACR-NCI Systems Biology : Confronting the Complexity of Cancer Conference in San Diego last week and presented two posters: Identification and Analysis of Essential Genes in Leukemic Cell Lines Using RNAi Screening and HT Screening for Bioactive Peptides that Increase Radiation Tolerance Using a Pooled Lentiviral Peptide Scanning Library. Although the poster session was relatively short for a small meeting, both posters were received well. Most of the interest was on the Identification of Essential Genes in Leukemic Cells poster as the shRNA library screening technique described in this study is generally applicable for validating pathways, identifying genes modulating cellular responses, and finding putative drug targets and biomarkers, which were the main interests of most of the attendees.

Almost all the talks at the conference were fascinating and many somewhat overwhelming in terms of the experimental work and data analysis involved. Researchers are struggling with the challenge to integrate and make sense of new experimental results in light of the massive amounts of expression profiling, protein-protein interaction, SNP analysis, genomic amplification, and other available profiling data for the range of genes, transcripts, proteins, and metabolites in a range of cell lines, tissues, and tumors. There exist numerous tools and approaches to synthesize and apply established results with new data but, clearly, much more work is necessary before a comprehensive and holistic understanding of the biological mechanisms controlling the many forms of cancer and tumorigenesis emerges.
 

Highlights and shRNA Library Screens

One of the highlights of the conference included the opening address from Stephen Friend of Sage Bionetworks during which he discussed top-down approaches to integrate these disparate data and ways to integrate and organize data sharing between various groups to develop more comprehensive mechanistic models. Also, Louis Staudt from the NCI talked about identification of a novel drug target for B-cell lymphoma using in vivo screening of mice with tet-regulated shRNA expression libraries. In another session, Michael Hemann from the MIT Koch Institute, used in vivo shRNA screening to investigate the mechanisms of various inhibitor drugs in vivo in the B-ALL Burkett’s lymphoma mouse model and, after starting with a pooled 2,200 shRNA expression library, was able to predict the mechanism of action for various compounds with just an 8-shRNA signature.

Unrelated to shRNA profiling, Jennifer Pietenpol from the Vanderbilt-Ingram Cancer Center gave an exceptional presentation of her thorough work genetically grouping, characterizing, and identifying driver genes in triple-negative breast cancers (ER-, progesterone-, and PR-) without HER2 amplification. In the same session, Valerie Weaver from UCSF discussed the often overlooked role of interactions between tumors and extracellular matrix proteins, and how this contributes to structural changes that lead to breast cancer tumor aggressiveness and pathology. In a different session, Ernest Fraenkel from the MIT presented work showing how information from protein-protein assays and expression profiles can be integrated using a Steiner Tree algorithm to work out functionally coherent pathways and aberrant interactions in EGFR networks in glioblastoma. Also, Todd Golub from the Broad Institute distilled vast amounts of expression profiles down to a list of 1,000 “landmark genes” that can be used to infer the expression levels of almost all other genes, so a complete expression profile can be generated that’s over 80% accurate just by measuring levels of 5% of the transcripts.
 

Software Tools and Resources

From a very practical perspective, many of the presenters highlighted numerous web-based software and database resources to assist in building, dissecting, and analyzing pathways. Paul Spellman from the Lawrence Berkeley National Laboratory discussed on-going work of The Cancer Genome Atlas (TCGA) Project that uses HT sequencing to genotype many dozens of cancer-derived cells lines across multiple cancer types. Andrea Califano from Columbia University discussed the use of the Master Regulator Analysis module of the Genomics Workbench ge(Workbench) to eludicate mechanisms of aberrant signal transduction in various cancer sub-types. Douglas Lauffenburger, also from MIT, discussed several cell network modeling approaches from simple relational to more structured logical and mechanistic with examples of how they could be used to analyze various type of data and pathways.

Other presenters referenced a variety of quite sophisticated tools and resources for cancer research and pathway analysis, links to some of these are listed below:
 

Paradigm—Pathway Analysis Software (UCSC)

ICGC— International Cancer Genome Consortium

ENCODE—Encyclopedia of DNA Elements (UCSC)

COSMIC—Catalog of Somatic Mutations in Cell Cancer (Sanger Center)

Biology Workbench— Database searching, analysis, and modeling tools (UCSC)

cBio Cancer Genomics Portal—Access to large-scale genomic cancer sets (MSKCC)

dbGap—Database of Genotypes and Phenotypes (NCBI)

ARACNe— Algorithm for the Reconstruction of Accurate Cellular Networks (Columbia)

MINDy— Modulator Inference by Network Dynamics to module modulator gene interference of a network (Columbia)

NetPhorest—Analysis for phosphorylation-dependent signaling motifs

NetWorKIN—Predicting in vivo kinase-substrate relationships

mFINDER—Network motifs detection tool (Weizmann Institute)

PTMScout— Analyze mass spec and other protein data for post-translational proteomics modifications
 

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Posted in Cancer, Conferences, Peptides, Software

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Cellecta’s DECIPHER Project RNAi Screening Tools at Roswell Park Cancer Institute

February 14th, 2011
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We have started this blog as a simple, unobtrusive conduit that enables us to talk directly to our customers, collaborators, and other interested researchers about new and interesting developments related to Cellecta’s technology and business. As the first post, we thought we would tell you about our recent agreement with the Roswell Park Cancer Institute (RPCI) that provides support to laboratories in their institution doing genome-wide RNAi screenings using our expanding portfolio of open-access DECIPHER™ pooled lentiviral shRNA expression libraries.

Although the plasmid versions of the DECIPHER shRNA library “modules” are available free-of-charge to any academic laboratory though the DECIPHER Project (www.decipherproject.net), the DECIPHER Technology Access and Maintenance (TAM) Program, which the RPCI just joined, lets them make this screening technology accessible through a well-supported centralized core facility. The DECIPHER TAM Program enables us to provide this institution with new modules of the packaged, ready-to-use shRNA expression libraries as they are developed, proactive support for all the labs using these resources, updates on new technological developments and protocols, and access to the latest software to analyze screening results.

Currently, as part of the open-access DECIPHER Project, there are 4 pooled shRNA library modules freely available—two modules targeting 10,000 human genes and two targeting 10,000 mouse genes. Each individual library module targets approximately 5,000 well-annotated genes with 27,500 shRNAs (5-6 shRNA target each transcript). Human and mouse 1 modules target that same set of genes related to signal transduction and cancer. The second modules target a broader range of genes that appear to be involved in disease processes and pathology but were not targeted in the first module. In a few weeks, we will release a human module 3 targeting 5,000 genes. Ultimately, there will be 5 modules targeting all human genes. As with all of our libraries, we utilize bar-coded inserts and have checked the quality of the DECIPHER modules using HT sequencing to ensure all shRNA sequences are present at sufficient levels to ensure comprehensive screening of the targeted gene set.

The ability to identify genes with specific functional activities makes screening with pooled shRNA expression libraries a very powerful technique to investigate the genetic controls regulating a wide range of biological responses. However, effective screening can be technically challenging. While the DECIPHER Project is helping to fulfill our goal to make basic tools for this type of analysis readily available to all labs, the DECIPHER TAM Program allows us to provide the resources and support that will empower labs to utilize these RNAi screening tools to their maximum potential. We are really looking forward to working with RPCI on this program.

More information about the DECIPHER Project can be found on www.decipherproject.net.

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Posted in Cancer, DECIPHER, Pooled shRNA Library, RNAi Screening, TAM Program

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