“When combined with the increasingly complete structural analyses of cancer genomes by the Cancer Genome Atlas and other such efforts, the experimental and analytical strategies for pooled shRNA screens described herein provide a feasible strategy to systematically identify the key genes involved in cancer initiation, maintenance, and progression and likely targets for therapeutic intervention,” the team concluded.
News Scan: Dec 12, 2008
CIRM Awards Tools and Technologies Grants
The California Institute for Regenerative Medicine this week announced that it will be funding 23 grants to 18 for-profit and not-for-profit institutions aimed at generating new tools and technologies to overcome barriers in stem cell research. At a meeting of the Independent Citizens’ Oversight Committee, the 29-member governing board for CIRM, members voted to approve more than $19 million in funding for this round of grants.
Among those institutions receiving CIRM grants were Invitrogen, which received $869,262 to develop disease models for neurodegenerative disorders; VistaGen, which received $971,558 to develop a human embryonic stem cell-based assay system for hepatocyte differentiation and predictive toxicology studies; and Vala Sciences, which received $906,629 to optimize the identification, selection, and induction of maturation of cardiomyocyte subtypes derived from hESCs.
The board voted to continue discussion of the 17 Tools and Technologies grants that the grants working group had placed in the second funding tier. These grants were recommended for funding only if funds permitted. The board will consider these additional grants at the next board meeting.
Leerink Swann Downgrades Life Science Tool Firms
Citing a survey that said that university-based researchers are seeing reductions in their budgets, investment bank Leerink Swann this week downgraded several life science tool firms that it covers.
Leerink Swann lowered its revenue estimates for Thermo Fisher Scientific, Millipore, PerkinElmer, and Sigma-Aldrich.
The investment bank said that a survey of 117 university-based life science researchers found that 68 percent of them are seeing reductions in their budgets. “While improved NIH funding looks to be a silver lining, we are particularly cautious on capital equipment trends,” Leerink Swann analyst Isaac Ro wrote in the report.
The bank said that based on its Washington contacts, consultants, and a panel it co-hosted earlier this week that included industry representatives and university researchers, current drafts of the stimulus bill call for a $1.2 billion to $1.7 billion increase in NIH funding for fiscal year 2009. That would represent a 4 percent to 6 percent increase year over year.
However, while NIH funding provides a vast majority of the funding for university-based research — around 75 percent by some estimates — Leerink Swann pointed to other pressures on life science tool makers. For example, university endowments, which provide the bulk of the remaining funding for academic labs, are likely seeing declines that would impact funding. In addition, private funding sources are likely facing similar cuts.
“Unfortunately, tighter budgets mean that certain research tools, even those that add value, will go by the wayside,” according to the report. “Not surprisingly, capital equipment was most often cited as the first thing to go. While we believe transformative technologies such as [next-generation sequencing] and genotyping arrays will continue to see good adoption, we believe basic lab equipment and high-end instrumentation will be challenged in the current environment.”
Leerink Swann said that it now expects Thermo Fisher Scientific to post a 2 percent decline in revenues for fiscal 2009 compared to fiscal 2008. It also expects Millipore to report a drop in revenue of 2 percent for 2009, PerkinElmer to report 1 percent revenue growth in 2009, and Sigma-Aldrich to report a revenue decline of 3 percent in FY2009. In all of these cases, it expects currency translation to have a negative effect.
Jackson ImmunoResearch Secures Non-Exclusive Rights to Thermo Fisher Fluorescent Dyes
Thermo Fisher Scientific announced this week that it has entered into a collaboration with Jackson ImmunoResearch Laboratories to facilitate the development of fluorescently labeled secondary antibodies for protein research and other applications.
Under the agreement, Jackson will have non-exclusive, global rights to use Thermo’s DyLight fluorescent dyes, which they plan to use in conjunction with Jackson’s secondary antibody line. The dyes are water-soluble, highly fluorescent, and photostable across a range of pH conditions.
Initially, Jackson plans to offer four of Thermo’s DyLight dyes conjugated to nearly 250 affinity-purified secondary antibodies, streptavidin, anti-biotin, anti-digoxin, anti-FITC, and purified immunoglobulins. The company is currently evaluating other DyLight dyes for potential use in its product line.
RNAi Consortium Uses shRNA Screen to Root Out Essential Cancer Genes
RNA interference-based screens are an effective way to delve into the functional underpinning of cancer cells, according to new research published by members of the RNAi Consortium.
The researchers, led by scientists at the Broad Institute, used RNAi screening with short hairpin RNAs to come up with a list of essential genes in a dozen cancer cell lines. They also used the screen to dig up a handful of genes involved in chronic myelogenous leukemia cell response to Novartis’ Gleevec (imatinib). The work is scheduled to appear online this week in the Proceedings of the National Academy of Sciences.
“We anticipate that systematic efforts to apply these approaches to study other cancer phenotypes will eventually lead to a more complete view of the Achilles’ heels of different types of cancers,” the authors wrote.
While projects such as The Cancer Genome Atlas and International Cancer Genome Consortium work towards characterizing the structural genetic changes behind various cancer types, the authors argued that there is also a need for corresponding studies into the associated functional changes in these cells.
In an effort to get at this functional information, the researchers applied a pooled screening approach developed by the RNAi Consortium, tapping into the consortium’s shRNA libraries. Overall, the libraries house roughly 170,000 shRNAs targeting 17,200 human genes and thousands more targeting 16,000 mouse genes.
But the team took advantage of a smaller sub-library that held 45,000 shRNAs targeting about 9,500 human genes, infecting various cell lines with the shRNA library and determining which shRNAs were over- and under-represented in surviving cells after a given amount of time. By assessing the genes targeted by these hairpin RNAs, the team was able to pick out genes whose expression influences survival under different conditions in different cell lines.
The team first tested the approach by screening Jurkat cells, a T lymphocyte cell line, to unearth genes involved in T cell resistance to apoptosis. Then, they screened a small cell lung cancer line to find genes conferring resistance to a compound called etoposide, which alters topoisomerase IIA activity. Because both screens pulled out plausible genes, the researchers were confident that their approach was feasible.
Next, the team turned their attention to 12 different cancer cells lines — representing everything from lung cancers to leukemias — to look for genes that were essential for survival in each. They screened the lines using ten or more rounds of infection with the shRNA library.
By amplifying shRNA sequences from surviving cells after roughly a month, digesting the hairpins with restriction enzymes, and using high-density, custom Affymetrix microarrays to measure the amount of these so-called half-hairpin barcodes, the researchers determined which shRNAs were more or less abundant in surviving cells.
The team then used unsupervised clustering and consensus clustering to group the various cell lines based on their shRNA abundance. They also came up with a new statistical score called the RNAi gene enrichment ranking to define essential genes based on the shRNA profiles detected in the cells.
Using this approach, the researchers found 268 “commonly essential genes” involved in pathways such as those for ribosomal proteins, mRNA processing, translation, and proteasome degradation. Known or suspected oncogenes, including KRAS, MYC, and MYB, were also among the top one percent of essential genes in one or more of the cell lines.
In addition, the team pinpointed genes that were specifically required in different types of cancer cells. For instance, they found 63 genes that were essential in four different non-small cell cancer cell lines and 32 genes that were essential in four different leukemia lines. The researchers also found instances of cell line-specific gene requirements in which certain genes were essential in just one of the 12 cancer cell lines.
By tweaking the approach slightly, the researchers were able to use the screen to find genes involved in imatinib response in a CML cell line. Imatinib, which is marketed as Gleevec by Novartis, inhibits a fusion protein that would otherwise keep a tyrosine kinase active in the cells, promoting cancer growth.
When they picked out and screened CML cells that were resistant to imatinib treatment, the researchers uncovered eight genes involved in imatinib response, providing new information about the pathways affected by the drug. And, they noted, RNAi screening may be useful for finding genes and pathways that interact with one another in various cell types and for fleshing out data from structural studies of cancer.