CH3 News

CH3 product most popular at tebu-bio!

Recently one of our European distributors, tebu-bio, tweeted:

Discover the most popular product on our website this week! http://t.co/A0AFg1Fhmr @CH3Bio #adenocarcinoma cell line

— tebu-bio (@tebubio) January 17, 2014

TUMORS, p53 AND SURVIVIN

HAPPY NEW YEAR!  FIRST POST OF 2013:

(click underlined text for links to references)

 A recent New York Times article published on December 22, 2012 described efforts to develop new drug compounds capable of targeting cancer cells that arise in many different organs of the human body.  Unfortunately, much was left unsaid with little source information to track down more specifics.  Fierce Biotech regurgitated the essentials of the article, but little in the way of basic science was offered.  Since this topic is of great interest here at CH3 BioSystems, some additional R&D context seems to be in order for the CH3 News web log.

The Times article suggests that the fatal liposarcoma that Mr. Bellino suffered from would have been an ideal test for a Sanofi drug developed to treat “…tumors that nearly always have the exact genetic problem the drug was meant to attack – a fusion of two large proteins.”  Although Gina Kolata did not explicitly name the fused proteins, the description brings to mind the chimeric TLS-CHOP oncoprotein (Rabbitts et al., 1993) identified in malignant liposarcoma.  TLS (Transformed in LipoSarcoma), known also as FUS (FUsed in Sarcoma), is an interesting methylarginine protein that is also implicated in the cause of a familial form of amyotrophic lateral sclerosis (Vance et al., 2009).

In 2011 a group in Japan presented data suggesting that the methylation of TLS activates transcription of survivin (Du et al., 2011), a member of the BIRC family of apoptosis inhibitory proteins that directly bind to and block caspases.

FIGURE 1. BIRC (Baculoviral Inhibitor of apoptosis Repeat Containing) Family of Apoptosis Inhibitory Proteins

So how are survivin and the arginine methylation of TLS biologically important for tumor growth? Why is this possibly relevant to fusion proteins, oncogenesis, protein-protein interactions, drug therapy and the article in the NY Times?  Consider the series of facts below:

• Survivin expression is repressed by normal, wild type p53 (Mita et al., 2008) 
• Mutation of p53 is frequently associated with solid tumors and is considered a likely reason for the abnormal apoptosis observed in tumors
• Thus, the cellular mechanism underlying p53 mutation-mediated tumor growth may be facilitated by aberrantly elevated levels of survivin expression
• Inactive p53 --> Elevated Survivin --> Inhibition of Apoptosis --> Increased tumor growth 

The NY Times article implied that half of all cancers exhibit p53 mutations and the other half involve p53 inactivation through the binding of p53 to another cellular protein.  Nutlins, drugs from Roche in Nutley NJ, and other drugs from Sanofi and Merck apparently can reverse the inactivation of p53 by driving a wedge between p53 and the protein bound inhibitor.

Alternatively, we think it’s worth proposing another drug target to reduce tumor growth based on the work of the Japanese team.  A concise summary of the team’s results from luciferase reporter assays is below:  

• TLS activates survivin transcription
• PRMT1, a protein arginine methlytransferase, activates survivin transcription
• TLS and PRMT1 synergistically co-activate transcription at the survivin promoter
• Catalytically-dead PRMT1 or treatment of cells with a methylation inhibitor both showed that the synergistic transcriptional activation is mediated by TLS arginine-methylation

 

 FIGURE 2. Synergistic TLS and PRMT1 co-activation of transcription at the survivin promoter

CONCLUSION:  Arginine methylation of TLS produces more active transcription of survivin.  Regardless of whether tumor growth is facilitated by mutation or inactivation of p53, or by the TLS-CHOP oncoprotein, the most logical approach for broad drug effectiveness would be to focus on the final common step of survivin transcription.  If pathologically high levels of survivin expression depend significantly on protein methylation, as suggested by the experiments reported above, a new comprehensive rationale for reducing tumor growth may be within reach.

By targeting protein arginine methyltransferase 1 (PRMT1) catalytic activity via treatment with inhibitors (Copeland et al., 2009), increased survivin expression can be reversed, tumor cell death re-activated and the size and viability of tumors will be reduced.

To make matters more complicated, of course, it should be pointed out that p53 is also regulated by arginine methylation (Jansson et al., 2008).  In this case the methylation appears to be carried out by PRMT5, a fundamentally different type of arginine methyltransferase.  We can try to sort that out later in future posts.

Send comments, criticisms, brain-storms and ideas about p53, survivin and the role of protein methylation in the biology of tumorigenesis to the CH3 News blog whether it is dogmatic, wild or just plain common sense. 

Anybody know more about the Sanofi and Merck drugs?

All feedback is welcome! 

 

 

 

 

 

 

Thanks to NYBA for hosting @ BIO INTERNATIONAL

CH3 BioSystems is grateful to the New York Biotechnology Association for providing us the opportunities we had at the One to One Partnering Meetings on the Exhibition floor at BIO International in Boston, MA in mid June. We are still working through all of the contacts and leads that were generated from the event.

 

 

 

Best wishes to all at The New York State Pavilion. Don't ever forget that the "Cure Start Here!"

An Intimation of Future Science Circa 2007

While doing some cyber housekeeping, we came across this first-time electronic effort to convey the excitement of Protein Methylation Detection Tools to the scientific community and the interested general public.  Although the logos and products have improved since this imageumentary was made back in 2007, the message remains the same today:

Protein Methylation is the next target-rich research area of biomedical knowledge innovation and first-in-class drug development for the new century of personalized medicine --- Methyl Marks Discovey!

 

 

Cancer, Epigenetics and Protein Arginine Methylation

Protein arginine methylation has been implicated in one way or another in 5 major disease categories: neurodevelopmental, autoimmune, cardiovascular, viral and neoplastic disease. The first blog posting in this series (PROTEIN Methylation; The Other Epigenetic Regulator) laid the theoretical groundwork for how protein arginine methylation might influence epigenetic molecular mechanisms that impact diverse targets and processes including genome stability, transcription factors, co-activators and co-repressors of gene expression.  Today’s post focuses in on how these protein methylation mechanisms may influence human cancers.  Several representative examples drawn from recent journal articles are offered for your review and comments.

Recently, estrogen receptor alpha (ERα) was identified as a target of protein arginine methyltransferase 1 (PRMT1).  Estradiol (E2) produces a rapid stimulation of the methylation of ERα in the DNA-binding domain of the receptor (Le Romancer et al., 2008).  The direct actions of the methylated form of ERα are thought to initiate a series of non-genomic interactions (Le Romancer et al., 2010).  Interestingly, though, these non-genomic actions have more far-reaching consequences.  Methylated ERα interacts with a cytoplasmic complex containing SRC, focal adhesion kinase (FAK) and the p85 sub-unit of phospoinositide-3-kinase (PI3K).  The end result of this signaling is activation of AKT, increased cell survival and cell proliferation, most likely due to nuclear integration of regulatory pathways that direct gene expression.

Another interesting finding in addition to the data on E2-induced methylation of ERα was the use of an antibody directed against methylated ERα in a series of human breast tumors (see Figure above).  Normal epithelial cells adjacent to the tumors exhibited only a weak reactivity and there was no reactivity of the antibody in myoepithelial cells.  A subset of the tumors (about half) displayed hypermethylated ERα.  Patient-dependent differences in methylation status could turn out to be very interesting in terms of both biological mechanisms and clinical relevance.

Vezzalini et al. used a broad-spectrum methylarginine-specific antibody to characterize methylation status in normal and tumor tissues.  The authors found that depending on the organ source of normal tissue, there are differences in the predominant subcellular location of protein methylarginine staining.  For example, methylarginine specific staining in endocrine tissues tends to be highest in nuclei, whereas intestinal epithelial cells have a cytoplasmic and Golgi-like pattern of staining.  Importantly, there is consistent staining from one normal specimen to another and staining is well correlated with immunostaining for PRMT1, the major protein arginine methyltransferase in mammalian cells.

Tumors, however, have a more heterogeneous staining from one specimen to the next with regard to intensity and sub-cellular location.  For instance, although normal thyroid gives a predominant nuclear stain, some papillary thyroid tumors instead can also display major cytoplasmic staining.  Table I (below) summarizes how very weak to negative methylarginine-reactivity is observed in a fraction of tumors analyzed.  Depending on tumor type, the reduced reactivity ranges from ~5% (breast) to ~30% (pancreatic duct).  The authors conclude that tumors may be stratified based on methylation patterns with regard to both staining intensity and subcellular location.

TABLE I

Lastly, two different tumor suppressor proteins are now known to be targets of protein arginine methylation that affects cellular biological responses.  BRCA1 is a multifaceted suppressor that is mutated in approximately half of all hereditary breast cancers.  Guendel et al. recently showed an association of BRCA1 with PRMT1.  They further showed that chemical inhibition of methylation or knockdown of PRMT1 decreases BRCA1 methylation and alters BRCA1 binding to promoter sites based on chromatin immunoprecipitation assays.  Interactions with specific transcription factors, such as SP1 and STAT1 could be either increased or decreased under conditions that reduce the methylation of BRCA1.  These results indicate that the methylation status of BRCA1 can influence transcription in a protein context-specific manner.

PDCD4 is another tumor suppressor protein that correlates well with better clinical outcomes when expressed at high levels particularly in lung, colon, ovarian and esophageal cancers.  There are, however, exceptions to this rule in breast tumors.  Some breast tumors that exhibit elevated levels of PDCD4 are associated with an unexpectedly poor clinical outcome.  The overexpression of the methyltransferase, PRMT5, in leukemia, lymphoma and gastric cancers encouraged Powers et al. to test whether PRMT5 might influence PDCD4 effects in tumor biology and growth.

For the assessment of tumor growth, the authors used an orthotopic NOD/SCID mouse transplantation model.  The MCF7e breast tumor cell line was stably transfected with PRMT5 and/or PDCD4.  Tumor growth in mice transplanted with empty vector control MCF7e cells, or cells over-expressing PRMT5, PDCD4 or both proteins was compared. Faster tumor growth and greater necrosis, edema and vascularization were observed in the transplants containing both proteins compared to vector, PRMT5, or PDCD4 alone transplants.

The authors of this study also examined clinical data from a large series of patient tumors with high expression of PDCD4 that were segregated into 4 categories based on PRMT5 expression levels (see Table II).  Those tumors with the highest levels of PRMT5 had the poorest outcomes, similar to a cohort of patients with low PDCD4.  When the levels of PRMT5 associated with tumors are lower, the probability of patient survival is increased in a statistically significant analysis.  Those tumors with high PDCD4 and the lowest levels of PRMT5 have a 20-year survival rate of 80% versus 43% for those patients in the top category with the relatively highest PRMT5 expression.

TABLE II

The precise understanding of the role of protein methylation in carcinogenesis clearly requires, and deserves, much greater study to enable advances in diagnosis and treatment.  However, based on the information currently available from animal and human studies on protein methylation, biomarkers based on protein methylation status may be useful in the future for determining clinical categories and prognosis.

Mitotic Bookmarking - More Epigenetic Control by Protein Arginine Methylation

The last posting at this site described the evidence for the role of protein arginine methylation as a key epigenetic regulator in addition to DNA methylation.  Since protein arginine methylation can produce important direct regulatory control over gene expression, it shouldn’t be surprising that it may also play a role in mitotic bookmarking, the latest epigenetic mechanism observed to regulate gene expression during normal cell development and carcinogenesis.  Mitotic bookmarking is a heritable form of epigenetic control that maintains cell identity following mitosis through a combination of histone modifications, DNA methylation and the retention of specific transcription factors at specific phenotypic promoter sites.  

An excellent review of bookmarking from Gary Stein’s lab at U. Mass. Cancer Center came out recently in Nature Reviews Genetics (Zaidi et al., 2010).  The Opinion piece does not directly reference protein arginine methylation, but it is intriguing that many of the effector molecules that carry out the bookmarking functions described in the review turn out to be methylarginine-modified proteins.  A few brief provocative examples are offered below to stimulate some brainstorming.  To understand the mechanism of the gene expression control exerted by bookmarking, it’s necessary to recognize that the compartmentalization and focal organization of protein complexes in nuclear microenvironments depends on diverse elements, such as RNA Pol I and Pol II, scaffold proteins, splicing factors and nuclear receptors.

Dynamic nuclear micro-domains tend to be organized around splicing factors (such as the serine/arginine rich (SR) proteins that process transcribed RNA) and scaffolding proteins (such as the Runt-related transcription factors, Runx) at specific sites of target-gene promoters to facilitate co-regulator recruitment.  These micro-processing domains are known to harbor PRMTs (protein arginine methyltransferases, Yanigida et al., 2004) and Runx1 transcriptional activity is regulated by arginine methylation mediated by PRMT1 (Zhao et al., 2008).  Krainer’s lab at Cold Spring Harbor recently reported on the potentially complex relationships between protein charge, methylation and the intracellular targeting and functions of a prototypical SR splicing protein (Sinha et al., 2010).

Zaidi et al. describe several gene bookmarking functions in development, lineage commitment and disease.  DNA sequence-specific protein associations, that exhibit accessibility to single strand nucleases in promoter regions only during mitosis, mark target genes for rapid transcription following mitosis.  For example, the single strand DNA binding protein, hnRNPK, can modulate transcription from the MYC promoter in this manner.  The basis for the preferential association/disassociation of hnRNPK from DNA is not clear, but perhaps the interaction depends on protein methylation state (Chen et al., 2008).  Similarly, Runx1 associates with Pol I transcribed ribosomal RNA (rRNA) genes and Pol II phenotype-specific genes involved in growth and differentiation, marking those genes for repression during early G1.

The recruitment of the methylarginine co-activator, CREB-binding protein (CBP), by specific transcription factors to the globin gene locus serves to enforce lineage-restricted expression of globin genes via rapid post-mitotic gene activation (Xin et al., 2007).  CCAAT/enhancer-binding proteins (C/EBPα, β, δ) also associate in a phenotype-specific way with mitotic chromosomes to mediate a program of growth and differentiation (Ali et al., 2008).  What role may be played by the arginine methylation of C/EBPβ (Kowen-Leutz et al., 2010) in lineage commitment and gene bookmarking, like the methylation of CBP, remains to be determined.

Alterations in gene bookmarking may also form the basis for some human diseases.  The association of the leukemogenic fusion protein, Runx1-ETO, with mitotic chromosomes up-regulates rRNA synthesis and may act as an epigenetic switch for unregulated cell proliferation (Bakshi et al., 2008).  Mixed lineage leukemia (MLL) protein, which is associated with the blood cell cancer, can also occupy mitotic chromatin to promote transcriptional reactivation after mitosis (Blobel et al., 2009).  Interestingly, there is a requirement for catalytically active PRMT1 in a mouse model of MLL-mediated leukemic transformation (Cheung et al., 2007).

The work on mitotic bookmarking is expanding our understanding of the increasing range of the epigenetic mechanisms that operate in biological systems.  Stay tuned for more links to how protein arginine methylation contributes to the epigenetics of mitotic bookmarks and methyl marks discovery!

Protein Methylation: The Other Epigenetic Regulator

Many thanks are due to Pete Jozsi and his excellent EpiGenie web site for introducing CH3 BioSystems to the Tweetogospheric world of sci-social media.  Observations, random ideas, topical reviews, bald speculations and various other brainstorms involving protein methylation will begin appearing here throughout 2011 and hopefully beyond.  To start things off, we are re-posting our first contribution to the EpiGenie web site into our very own electron-rich user interface (see below).

Please comment, and remember to visit EpiGenie for updates on all things epigenetic!

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Epigenetics is the study of heritable changes of phenotype that occur without alteration of the genome’s nucleotide sequence.  Epigenetic regulation is too often viewed as DNA modification, typically methylation, or chromatin remodeling by ATP-dependent protein complexes.  But, besides DNA methylation and chromatin reorganization, it turns out that the post-translational modification of proteins is also extremely important for gene expression.  In particular, the rapidly expanding field of protein arginine methylation is noteworthy in several instances for affecting gene expression through the placement of methyl marks on many different proteins in addition to the histones (Lee and Stallcup, 2009).

 

There may be rather broad relevance of this post-translational modification for human biology ranging from inherited dietary preferences to carcinogenesis.  As long ago as 2000, the Stallcup lab at USC discovered that protein arginine methyltransferases (PRMTs) serve as co-activators.  The substrates for methylation in the early studies were found to be histone proteins, but since then everything from DNA binders, elongation factors, signaling molecules and many other proteins affecting gene expression exhibit altered functions and/or cellular locations as a result of arginine methylation.

For example, long-term changes in offspring phenotype are associated with dietary restrictions (Kappeler and Meaney, 2010) and reduced caloric intake (Vaquero and Reinberg, 2009).  The precise mechanism(s) of the epigenetic modifications observed are unresolved, but clearly are secondary to changes in the gene expression of metabolic intermediates.  Of course, DNA methylation and differential access of chromatin remodelers based on histone methylation have been considered, but arginine methylation of other nuclear proteins may also play a role.  Many well-known transcriptional co-regulators (e.g. CBP/p300, steroid receptor co-activator/p160, PPAR co-activator γ 1α  and others) are substrates for PRMTs.  Recently the arginine methylation of C/EBPβ, a transcription factor that regulates genes involved in metabolism, was found to regulate the interaction of C/EBPβ with epigenetic gene regulatory protein complexes during cell differentiation (Kowenz-Leutz et al., 2010).  DNA methylation-mediated silencing actions may further be regulated by arginine methylation of MBD2, a methyl-DNA binding protein (Tan and Nakielny, 2006).  The methylation of MBD2 decreases binding to methyl-DNA and histone deacetylases, thus decreasing transcriptional repression.

The fidelity of genetic inheritance is fundamentally dependent on proper germ cell development.  Here again, current leading edge research casts a spotlight on PRMTs and the substrates upon which methyl marks are placed.  The enzymes and methylproteins are emerging as critically important molecular determinants of proper germ cell development.  Recent examples are the arginine methylation of the RNA helicase, vasa and its vertebrate homologs (Kirino et al., 2010), and piwi proteins, which suppress mobile genetic elements in the germ cells of multi-cellular animals (Vagin et al., 2009).

Lastly, we have the example of cell signaling via arginine methylated estrogen receptor α  (ERα).  The methylation of ERa is required for the extra-nuclear function of the receptor to signal to downstream Src/FAK and p85/Akt for proliferative and survival actions.  Additional immunohistochemical data from a cohort of breast cancer patients also provide implications for potential epigenetic mechanisms of tumorigenesis (Le Romancer et al., 2010).

These few examples just barely scratch the surface of the potential involvement of protein arginine methylation in the epigenetics of human health and disease.  If you know of other examples, have comments or questions, please post any input here.  CH3 will welcome any opportunity to spread interest and further understanding of protein arginine methylation and its relationships to biology and biomedical science.

5 Reasons Why BIO International 2010 in Chicago Rocks!

We are having a most awesome time in Chicago. Here are five reasons why:

1) Our lodgings. Being a startup with a lean budget (not to say cheap) we let Hotwire find our hotel room. Hotwire did not let us down. The Chicago South Loop Hotel has been a very nice home away from home within walking distance (or an easy cab ride) from the convention center. 

2) Making connections with people face to face, people we know only through the literature or journal articles, is priceless. The Bio Convention organizers have done an amazing job making this behemoth of a convention run smoothly from registration to meeting scheduling.

3) The global aspect to the meeting brings home the fact that Science and commerce these days is totally a small world thing. Science, like math, is a universal language without borders or (in its purest form) politics.

4) Chicago herself is a gracious host city. How she managed the most gorgeous weather in North America I'll never know. But we're grateful.

5) The cherry on top! Dr. John Aletta was visiting the Novo Nordisk booth in the Exhibition Hall when he was encouraged to enter a drawing. On the spot he won a brand new Apple iPad!!!

And this is only Day One of the meeting!

The CERN Large Hadron Colider Rap!

CH3 will see you at BIO International this year!

Since CH3's inception we've heard about the BIO International Convention, a mega-event for biotechnology. Our advisers said this is one trade show we did not want to miss. Together with many large and small businesses across the state of New York and the Center of Excellence in Bioinformatics & Life Sciences, we will be attending this year! Here's a video to get a taste of what the 2010 meeting in Chicago is all about:

Digg CH3 on Twitter!

Yup, CH3 has joined the river of information, news, social and business networking that is Twitter. You'll find our tweets in the sidebar here but if you'd like to visit the CH3Bio Twitter feed go to twitter.com/CH3Bio.

Twitter is fresh and stimulating. We'll be tweeting and posting all kinds of stuff; wise and funny quotes from scientists past and present, retweets from science journalists and fellow entrepreneurs around the world. We will focus on news that furthers all Science, including our own uniquely inventive research and business. We won't flood you with a gazillion tweets an hour, except maybe the first week of May. That's when we'll be at the BIO International meeting in Chicago. You can expect lots of tweets out of Chicago as we promote not only CH3 but all New York State biotechnology, from start ups to Big Pharma. It should be crazy, busy and fun!

CH3 BioSystems Wins Entrepreneur Award

CH3 BioSystems LLC wins First Prize in the Fourth Annual Henry A. Panasci Jr. Technology Entrepreneurship Competition.

Biotech in Western New York

Genetic Engineering & Biotechnology News article features biotechnology in Buffalo.

Entrepreneurial Initiatives Conference

CH3 BioSystems participates in fuse2007, “Entrepreneurial Initiatives - Emerging Upstate Companies-Future Growth and Profitability.” Video link, Whitman School of Management, Syracuse University.

CH3 BioSystems Promotes Cutting Edge Collaborations

CH3 BioSystems promotes cutting edge collaborations with academia. Partnerships with Buffalo Niagara Area institutions aimed at fostering discovery in the realm of Molecular Recognition in Biological Systems offer a valuable medium to grow the field of protein methylation biology.

CH3 Products Featured in Major BioTech Industry Trade Journals

CH3 BioSystems Anti-mRG methylarginine-specific antibody featured on New Products page in Drug Discovery and Development and Genetic Engineering News.

CH3 BioSystems Featured in Business First Buffalo

First published in Business First February 6, 2009

More than a dozen companies are taking advantage of lab space, shared services and prime offices in the heart of the Buffalo Niagara Medical Campus.

They have done so by co-locating at the University at Buffalo Center of Excellence in Bioinformatics and Life Sciences.

The facility has become a hotbed for collaboration and research, says Marnie LaVigne, director of business development at the center. The building currently houses about 300 people, with nearly a third working for private sector firms.

Since its inception in 2001, the Center of Excellence has worked with over 50 companies, providing research and development support for technology development projects, and subsidizing office and lab space. It has received over $2 million in grants over the past 18 months to help develop a high tech workforce in the region. This year, the company expects to work with 15 companies.

“It’s exciting. It’s really all kind of coming together that we’re working together as a community,” LaVigne says.

Following are short profiles of five current Center of Excellence tenants.

CH3 BioSystems (www.ch3biosystems.com) was created in 2007 after winning the Henry A. Panasci Jr. Technology Entrepreneurship Competition at UB. The company, which opened offices at the Center of Excellence this summer, is a biopharmaceutical supplier of molecular tools and services for biomedical researchers. It has since received funding through UB’s Center of Advanced Technology through the State Office of Science Technology and Academic Research (NYSTAR).