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Muscular dystrophy has deficient stem cell dystrophin

Posted in Biological Networks, Ca2+ triggered activation, Ca2+ triggered activation, Cell Biology, Signaling & Cell Circuits, Clinical Genomics, Curation, Developmental biology, Disease Biology, Gene Regulation, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Inflammasome, Interviews with Scientific Leaders, Investment in Technological Breakthrough, MS, Neurodegenerative Diseases, Neurohumoral Transmission, Neurological Diseases, Neuroscience, Pharmaceutical Analytics, Pharmaceutical Discovery, Population genetics, Proteins, Proteomics, Regenerative Biology and Medicine, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Skeletal muscle regeneration, Stem Cells for Regenerative Medicine, tagged blocking cell differentiation, Ca2+-regulation, Clinical trial, dystrophin, genomic expression, Muscular dystrophy, skeletal and cardiomyocyte, Stem cell on November 21, 2015| Leave a Comment »

Muscular dystrophy has deficient stem cell dystrophin

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Article ID #198: Muscular dystrophy has deficient stem cell dystrophin. Published on 11/21/2015

WordCloud Image Produced by Adam Tubman

Dystrophin Deficient Stem Cell Pathology

Muscular Dystrophy is a Stem Cell-Based Disease

November 19, 2015 by mburatov

https://beyondthedish.wordpress.com/2015/11/19/muscular-dystrophy-is-a-stem-cell-based-disease/

Michael Rudnicki, who has done pioneering work in muscle stem cell biology and muscle regeneration, and whose work has been featured several times on this blog, has struck again. Rudnicki, who serves as director of the Regenerative Medicine Program at The Ottawa Hospital and a professor at the University of Ottawa and holds the prestigious Canada Research Chair in Molecular Genetics, teamed up with workers from the Sprott Centre for Stem Cell Research and the Sinclair Centre for Regenerative Medicine to investigate the role of muscle-specific stem cells in patients who suffer from Duchenne muscular dystrophy. This new earth-shaking study, which was published in the journal Nature Medicine (November 16, 2015), has changed the way we think about muscular dystrophy and will almost certainly force people to rethink the treatments and cures for this dreadful disease.

According to this new study, Duchenne muscular dystrophy directly affects muscle stem cells, and is, largely a disease of muscle stem cells.

Rudicki said: “For nearly 20 years, we’ve thought that the muscle weakness observed in patients with Duchenne muscular dystrophy is primarily due to problems in their muscle fibers, but our research shows that it is also due to intrinsic defects in the function of their muscle stem cells. This completely changes our understanding of Duchenne muscular dystrophy and could eventually lead to far more effective treatments.”

Muscular dystrophy comes in several different forms, but the predominant sign of muscular dystrophy is progressive muscle weakness. Altogether, muscular dystrophy refers to a group of more than 30 genetic diseases, all of which cause progressive weakness and degeneration of skeletal muscles used during voluntary movement. Approximately half of all who suffer from muscular dystrophy have Duchenne muscular dystrophy (DMD). Because muscular dystrophy results from mutations in the dystrophin gene, which is on the X chromosome, the vast majority of muscular dystrophy patients are male. Girls can be carriers of muscular dystrophy and can be mildly affected.

Interestingly, somewhere around one-third of boys who suffer from DMD have no family history of the disease. Because the dystrophin gene is so large, spontaneous mutations in it are probably relatively common.

The signs and symptoms typically appear between the ages of 2 and 3, and may include frequent falls, difficulty getting up from a lying or sitting position, trouble running and jumping, a strange, shuffling way of walking or having a tendency to walk on their toes, calf muscles that are abnormally large, muscle pain and stiffness, and some learning disabilities.

Muscular dystrophy affects all ethnic groups and occurs globally. It affects around 1 in every 3,500 to 6,000 male births each year in the United States. DMD affects approximately one in 3,600 boys.

Because DMD results from mutations in the dystrophin gene, the vast majority of muscular dystrophy research was based on a simple model in which the Dystrophin protein played a structural role in the structural integrity of muscle fibers. Abnormal versions of the Dystrophin protein caused the muscle fibers to become damaged and die as a result of contraction. Dystrophin anchors the cytoskeleton of the muscle fibers, which are essential for muscle contraction, to the muscle cell membrane, and then to the extracellular matrix outside the cell that serves as a foundation upon which the muscle cells are built.

However in this current study, Rudnicki and his team discovered that muscle stem cells also express the dystrophin protein. This is a revelation because Dystrophin was thought to be protein that ONLY appeared in mature muscle. However, in this study, it became exceedingly clear that in the absence of Dystrophin, muscle stem cells generated ten-fold fewer muscle precursor cells, and, consequently, far fewer functional muscle fibers. Dystrophin is also a component of a signal transduction pathway that allows muscle stem cells to properly ascertain if they need to replace dead or dying muscle. Muscle stem cells repair the muscle in response to injury or exercise by dividing to generate precursor cells that differentiate into muscle fibers.

Even though Rudnicki used mice as a model system in these experiments, the Dystrophin protein is highly conserved in most vertebrate animals. Therefore, it is highly likely that these results will also apply to human muscle stem cells.

Gene therapy experiments and trials are in progress and even show some promise, but Rudnicki’s work tells us that gene therapy approaches must target muscle stem cells as well as muscle fibers if they are to work properly.

“We’re already looking at approaches to correct this problem in muscle stem cells,” said Dr. Rudnicki.

This paper has received high praise from the likes of Ronald Worton, who was one of the co-discovers of the dystrophin gene with Louis Kunkel in 1987.

Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells

Emi Shoji, Hidetoshi Sakurai, Tokiko Nishino, Tatsutoshi Nakahata, Toshio Heike, Tomonari Awaya, Nobuharu Fujii, Yasuko Manabe, Masafumi Matsuo & Atsuko Sehara-Fujisawa

Scientific Reports 5, Article number: 12831 (2015) http://dx.doi.org:/10.1038/srep12831

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca²⁺) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca²⁺ overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs.

Duchenne muscular dystrophy (DMD) is characterised by progressive muscle atrophy and weakness that eventually leads to ambulatory and respiratory deficiency from early childhood¹. It is an X-linked recessive inherited disease with a relatively high frequency of 1 in 3500 males^1,2.DMD, which is responsible for DMD, encodes 79 exons and produces dystrophin, which is one of the largest known cytoskeletal structural proteins³. Most DMD patients have various types of deletions or mutations in DMD that create premature terminations, resulting in a loss of protein expression⁴. Several promising approaches could be used to treat this devastating disease, such as mutation-specific drug exon-skipping^5,6, cell therapy⁷, and gene therapy^1,2.

Myoblasts from patients are the most common cell sources for assessing the disease phenotypes of DMD^11,12. …Previous reports have shown that muscle cell differentiation from DMD patient myoblasts is delayed and that these cells have poor proliferation capacity compared to those of healthy individuals^11,12. Our study revealed that control and DMD myoblasts obtained by activating tetracycline-dependent MyoD transfected into iPS cells (iPS^tet-MyoD cells) have comparable growth and differentiation potential and can produce a large number of intact and homogeneous myotubes repeatedly.

The pathogenesis of DMD is initiated and progresses with muscle contraction. The degree of muscle cell damage at the early stage of DMD can be evaluated by measuring the leakage of creatine kinase (CK) into the extracellular space¹⁵. Excess calcium ion (Ca²⁺) influx into skeletal muscle cells, together with increased susceptibility to plasma membrane injury, is regarded as the initial trigger of muscle damage in DMD^{19,20,21,22,23,24}. Targeting these early pathogenic events is considered essential for developing therapeutics for DMD.

In this study, we established a novel evaluation system to analyse the cellular basis of early DMD pathogenesis by comparing DMD myotubes with the same clone but with truncated dystrophin-expressing DMD myotubes, using the exon-skipping technique. We demonstrated through in vitro contraction that excessive Ca²⁺ influx is one of the earliest events to occur in intact dystrophin-deficient muscle leading to extracellular leakage of CK in DMD myotubes.

Generation of tetracycline-inducible MyoD-transfected DMD patient-derived iPSCs (iPS^tet-MyoD cells)

Figure 1: Generation and characterization of control and DMD patient-derived Tet-MyoD-transfected hiPS cells. Full size image

Morphologically and physiologically comparable intact myotubes differentiated from control and DMD-derived hiPSCs

Figure 2: Morphologically and physiologically comparable skeletal muscle cells differentiated from Control-iPS^tet-MyoD and DMD-iPS^tet-MyoD. Full size image

Exon-skipping with AO88 restored expression of Dystrophin in DMD myotubes differentiated from DMD-iPS^tet-MyoD cells

Figure 3: Restoration of dystrophin protein expression by AO88. Full size image

Restored dystrophin expression attenuates Ca²⁺ overflow in DMD-Myocytes

Figure 4: Restored expression of dystrophin diminishes Ca²⁺ influx in DMD muscle in response to electric stimulation. Full size image

Ca²⁺ influx provokes skeletal muscle cellular damage in DMD muscle

Figure 5: Ca²⁺ influx induces prominent skeletal muscle cellular damage in DMD-Myocytes. Full size image

Skeletal muscle differentiation in myoblasts from DMD patients is generally delayed compared to that in healthy individuals^11,36,37. Our differentiation system successfully induced the formation of myotubes from DMD patients, and the myotubes displayed analogous morphology and maturity compared with control myotubes (Fig. 2a–c). Comparing myotubes generated from patient-derived iPS cells with those derived from the same DMD clones but expressing dystrophin by application of the exon-skipping technique enabled us to demonstrate the primary cellular phenotypes in skeletal muscle solely resulting from the loss of the dystrophin protein (Fig. 4b). Our results demonstrate that truncated but functional dystrophin protein expression improved the cellular phenotype of DMD myotubes.

In DMD, the lack of dystrophin induces an excess influx of Ca²⁺ , leading to pathological dystrophic changes²². We consistently observed excess Ca²⁺ influx in DMD-Myocytes compared to Control-Myocytes (Supplementary Figure S3a and S3b) in response to electric stimulation. TRP channels, which are mechanical stimuli-activated Ca²⁺ channels⁴⁰that are expressed in skeletal muscle cells⁴¹, can account for this pathogenic Ca²⁺ influx…

In conclusion, our study revealed that the absence of dystrophin protein induces skeletal muscle damage by allowing excess Ca²⁺ influx in DMD myotubes. Our experimental system recapitulated the early phase of DMD pathology as demonstrated by visualisation and quantification of Ca²⁺ influx using intact myotubes differentiated from hiPS cells. This evaluation system significantly expands prospective applications with regard to assessing the effectiveness of exon-skipping drugs and also enables the discovery of drugs that regulate the initial events in DMD.

Duchenne muscular dystrophy affects stem cells, University of Ottawa study finds

New treatments could one day be available for the most common form of muscular dystrophy after a study suggests the debilitating genetic disease affects the stem cells that produce healthy muscle fibres.

The findings are based on research from the University of Ottawa and The Ottawa Hospital, published Monday in the journal Nature Medicine.

For nearly two decades, doctors had thought the muscular weakness that is the hallmark of the disease was due to problems with human muscle fibers, said Dr. Michael Rudnicki, the study’s senior author.

The new research shows the specific protein characterized by its absence in Duchenne muscular dystrophy normally exists in stem cells.

Dystrophin protein found in stem cells

“The prevailing notion was that the protein that’s missing in Duchenne muscular dystrophy — a protein called dystrophin — was not involved at all in the function of the stem cells.”

http://soundcloud.com/cbcottawa1

When the genetic mutations caused by Duchenne muscular dystrophy inhibit the production of dystrophin in stem cells, those stem cells produce significantly fewer precursor cells — and thus fewer properly functioning muscle fibres. Further, stem cells need dystrophin to sense their environment to figure out if they need to divide to produce more stem cells or perform muscle repair work.

Genetic repair might treat Duchenne muscular dystrophy

July 25, 2011|By Thomas H. Maugh II, Los Angeles Times

A genetic technique that allows the body to work around a crucial mutation that causes Duchenne muscular dystrophy increased the mass and function of muscles in a small group of patients with the devastating disease, paving the way for larger clinical trials of the drug. The study in a handful of boys age 5 to 15 showed that patients receiving the highest level of the drug, called AVI-4658 or eteplirsen, had a significant increase in production of a missing protein and increases in muscle fibers. The study demonstrated that the drug is safe in the short term. Results were reported Sunday in the journal Lancet.

Duchenne muscular dystrophy affects about one in every 3,500 males worldwide. It is caused by any one of several different mutations that affect production of a protein called dystrophin, which is important for the production and maintenance of muscle fibers. Affected patients become unable to walk and must use a wheelchair by age 8 to 12. Deterioration continues through their teens and 20s, and the condition typically proves fatal as muscle failure impairs their ability to breathe.

This study is designed to assess the efficacy, safety, tolerability, and pharmacokinetics (PK) of AVI-4658 (eteplirsen) in both 50.0 mg/kg and 30.0 mg/kg doses administered over 24 weeks in subjects diagnosed with Duchenne muscular dystrophy (DMD).

Condition	Intervention	Phase
Duchenne Muscular Dystrophy	Drug: AVI-4658 (Eteplirsen) Other: Placebo	Phase 2

Study Type:	Interventional
Study Design:	Allocation: Randomized Endpoint Classification: Safety/Efficacy Study Intervention Model: Parallel Assignment Masking: Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor) Primary Purpose: Treatment
Official Title:	A Randomized, Double-Blind, Placebo-Controlled, Multiple Dose Efficacy, Safety, Tolerability and Pharmacokinetics Study of AVI-4658(Eteplirsen),in the Treatment of Ambulant Subjects With Duchenne Muscular Dystrophy

Resource links provided by NLM:

Genetics Home Reference related topics: Duchenne and Becker muscular dystrophy

MedlinePlus related topics: Muscular Dystrophy

Genetic and Rare Diseases Information Center resources: Becker Muscular Dystrophy Duchenne Muscular Dystrophy Muscular Dystrophy

U.S. FDA Resources

Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

Nicolas A Dumont, Yu Xin Wang, Julia von Maltzahn, Alessandra Pasut, C Florian Bentzinger, Caroline E Brun & Michael A Rudnicki

Nature Medicine(2015) http://dx.doi.org:/10.1038/nm.3990

Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.

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Telomere length

Posted in Cell Biology, Chemical Genetics, Curation, Developmental biology, Gene Regulation, Proteomics, tagged enzymatic shortening, telomer length on November 21, 2015| Leave a Comment »

Telomere length

Larry H. Bernstein, MD, FCAP, Curator

LPBI

New Enzyme Discovered for Sustaining Telomere Length

http://www.genengnews.com/gen-news-highlights/new-enzyme-discovered-for-sustaining-telomere-length/81251981/

http://www.genengnews.com/Media/images/GENHighlight/Picture18124516425.jpg

Researchers developed a novel assay to identify telomere length regulators and showed that ATM inhibition shortens telomeres, whereas ATM activation elongates telomeres. [Lee et al., 2015, Cell Reports 13, 1–10]

In the early years of molecular biology research, scientists studying chromosomal structure and composition noticed that the terminal ends of chromosomes, called telomeres, would gradually become shorter with each successive round of cellular replication. This process would continue until the chromosome reached a certain length, ultimately becoming unstable and causing the cell to die. Conversely, the scientists noticed that for certain genetic disorders, such as cancer, an abnormally long telomere length led to genome anomalies that were closely associated with the cancer phenotype.

In 1984, researchers Elizabeth Blackburn, Ph.D., and Carol Greider, Ph.D., who was at the time a graduate student in Dr. Blackburn’s laboratory, discovered the telomerase enzyme, which was responsible for maintaining the appropriate length of telomerase after chromosomal replication. Drs. Blackburn and Greider would go on to be awarded the 2009 Nobel Prize in Physiology and Medicine, along with Jack Szostak, Ph.D. for their work on molecular mechanisms of the telomerase enzyme.

Yet, even during their seminal work, the investigators quickly realized that other molecules besides telomerase must be involved in maintaining the protective caps at the end of chromosomes. Now, researchers at Johns Hopkins report uncovering the role of an additional enzyme crucial to telomere length and say the novel method they could be used to speed discovery of other proteins and processes that are involved in telomere stability.

“We’ve known for a long time that telomerase doesn’t tell the whole story of why chromosomes’ telomeres are a given length, but with the tools we had, it was difficult to figure out which proteins were responsible for getting telomerase to do its work,” explained Dr. Greider, professor, and director of molecular biology and genetics in the Johns Hopkins Institute for Basic Biomedical Sciences.

The findings from this study were published recently in Cell Reports through an article entitled “ATM Kinase Is Required for Telomere Elongation in Mouse and Human Cells.”

Understanding the mechanisms that are needed to lengthen telomeres has broad health implications, since shortened telomeres have been implicated in aging and diseases as diverse as lung and bone marrow disorders, while overly long telomeres are linked to cancer. Cells need a well-tuned process to keep adding the right number of building blocks back onto telomeres over an organism’s lifetime.

Unfortunately, until recently, the methods researchers used to study telomere length were extremely time-consuming, often taking months of work to study cells grown in vitro, searching for detectable differences in telomere length. However, Dr. Greider’s team developed a new tool for measuring telomere length in yeast. The idea was to artificially cut mammalian cells’ telomeres and then detect elongation by telomerase—a test that would take less than a day, and could be performed even if the blocked proteins were needed for cells to divide.

The new test, dubbed addition of de novo initiated telomeres (ADDIT) was used to observe an enzyme long suspected to be involved in telomere maintenance, ATM kinase. “ATM kinase was known to be involved in DNA repair, but there were conflicting reports about whether it had a role in telomere lengthening,” noted Dr. Greider.

The Hopkins researchers blocked the enzyme in lab-grown mouse cells and used ADDIT to find that it was indeed needed to lengthen telomeres. They confirmed their result by using the old, three-month-long telomere test, which lead to the same outcome.

Additionally, the team also found that in normal mouse cells, a drug that blocks an enzyme called PARP1 would activate ATM kinase and spur telomere lengthening. This finding has the potential to impact drug-based telomere elongation for treating short-telomere diseases, such as bone marrow failure.

Dr. Greider and her team were excited by their findings and plan to use ADDIT to find out more about the telomere-lengthening biochemical pathway that ATM kinase participates.

“The potential applications are very exciting,” stated lead author Stella Lee, Ph.D., postdoctoral fellow in Dr. Greider’s laboratory. “Ultimately ADDIT can help us understand how cells strike a balance between aging and the uncontrolled cell growth of cancer, which is very intriguing.”

Nobel Laureate Blackburn Named Salk Institute’s New President

Elizabeth H. Blackburn, Ph.D., a 2009 Nobel laureate who has specialized in telomere and telomerase research, was named the new president of the Salk Institute for Biological Studies today, effective January 1, 2016.

“The Salk is full of absolutely terrific people and brimming with great science. Building on its distinguished history and current success, I am delighted to be playing a role in continuing and growing its major contributions to science and health research,” Dr. Blackburn said in a statement. “I am truly honored to be asked to be the next president of the Salk Institute.”

Dr. Blackburn has been a non-resident fellow at the institute since 2001, where she has been one of a group of investigators that advise the institute’s leadership and play key decision-making roles in appointing and promoting Salk professors. In addition, she has been the Morris Herzstein Professor of Biology and Physiology in the department of biochemistry and biophysics at the University of California San Francisco (UCSF).

In 2009, she was named one of three co-winners of the Nobel Prize in Physiology or Medicine “for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.” Dr. Blackburn discovered the molecular nature of telomeres—the ends of eukaryotic chromosomes that serve as protective caps essential for preserving the genetic information—and co-discovered the ribonucleoprotein enzyme telomerase.

Those discoveries helped launch new research around telomeres and telomerase, both believed to play central roles in aging and diseases that include cancer.

In addition to the Nobel Prize, Dr. Blackburn has received nearly every major award in science, including the Lasker, Gruber, and Gairdner prizes. In 2007, she was named to the TIME 100 yearly list of the world’s most influential people. Dr. Blackburn is also a member of the National Academy of Sciences, the National Academy of Medicine, and the Royal Society of London.

Dr. Blackburn was born in Hobart, Tasmania, Australia, to a family of doctors and scientists. Her parents were both family physicians, while her grandfather and great-grandfather were geologists.

Inspired by her fascination with animals and a biography of Marie Curie, Dr. Blackburn chose to also pursue a career in science. She earned her B.Sc. degree in 1970 and her M.Sc. degree in biochemistry, both from the University of Melbourne. She earned her Ph.D. in molecular biology from the University of Cambridge in 1975, then conducted postdoctoral research in molecular and cellular biology at Yale University from 1975 to 1977.

ATM Kinase Is Required for Telomere Elongation in Mouse and Human Cells

Stella Suyong Lee, Craig Bohrson, Alexandra Mims Pike, Sarah Jo Wheelan, Carol Widney Greider

DOI: http://dx.doi.org/10.1016/j.celrep.2015.10.035

•ADDIT assay measures telomerase-mediated addition at a single telomere
•De novo telomere addition in mouse cells requires ATM kinase
•ATM inhibition blocks bulk telomere elongation in both mouse and human cells
•Excess activation of ATM by inhibition of PARP1 increases telomere addition

Short telomeres induce a DNA damage response, senescence, and apoptosis, thus maintaining telomere length equilibrium is essential for cell viability. Telomerase addition of telomere repeats is tightly regulated in cells. To probe pathways that regulate telomere addition, we developed the ADDIT assay to measure new telomere addition at a single telomere in vivo. Sequence analysis showed telomerase-specific addition of repeats onto a new telomere occurred in just 48 hr. Using the ADDIT assay, we found that ATM is required for addition of new repeats onto telomeres in mouse cells. Evaluation of bulk telomeres, in both human and mouse cells, showed that blocking ATM inhibited telomere elongation. Finally, the activation of ATM through the inhibition of PARP1 resulted in increased telomere elongation, supporting the central role of the ATM pathway in regulating telomere addition. Understanding this role of ATM may yield new areas for possible therapeutic intervention in telomere-mediated disease.

Vertebrate telomeres are repetitive TTAGGG DNA sequences located at the ends of chromosomes, which protect the coding regions of DNA. In mammalian germline cells and ∼85% of cancers, telomere length is maintained by the dimeric ribonucleoprotein telomerase, which catalyzes the addition of TTAGGG repeats to counteract telomere shortening and cellular senescence (Shay and Bacchetti, 1997, Kim et al., 1994, Wenz et al., 2001). The minimal catalytic core of human telomerase consists of the telomerase reverse transcriptase protein (hTERT), telomerase RNA (hTR), and the protein dyskerin (Cohen et al., 2007).

The differentiation of telomeres from broken chromosome ends is conferred by a family of six telomere-specific binding proteins collectively termed “shelterin” (de Lange, 2005). This complex consists of the double-stranded binding proteins TRF1 and TRF2, the single-stranded binding proteins POT1 and TPP1, the bridging protein TIN2 that links these two groups of proteins, and Rap1 (reviewed in Palm and de Lange, 2008). TRF1 protects the telomere and negatively regulates telomerase-mediated telomere lengthening (van Steensel and de Lange, 1997, Smogorzewska et al., 2000, Ancelin et al., 2002, Karlseder et al., 2002). TRF1 also facilitates the progression of the replication machinery; deletion of TRF1 increases replication fork stalling, leading to ATR kinase activation and a “fragile telomere” phenotype (Sfeir et al., 2009, Martínez et al., 2009). The TRF1-mediated repression of the ATR response requires recruitment of the shelterin components TIN2 and the TPP1/POT1 heterodimer (Zimmermann et al., 2014).

TPP1 and POT1 also have roles in mediating telomere-length regulation. A surface on the N-terminal oligonucleotide/oligosaccharide-binding (OB) domain of TPP1 termed the TEL patch activates telomerase by stimulating telomerase processivity and providing a direct binding site for telomerase recruitment to telomeres; mutation of the TEL patch can lead to telomere shortening syndromes characterized by bone marrow failure (Abreu et al., 2010, Nandakumar et al., 2012, Zhong et al., 2012, Kocak et al., 2014, Guo et al., 2014, Dalby et al., 2015). Additionally, mutation analyses at sites independent of the TEL patch have implicated TPP1 as part of a telomere-length-dependent feedback loop that regulates telomere-length homeostasis (Sexton et al., 2014). A mutant form of POT1 that abrogates binding to single-stranded DNA (POT1ΔOB) deregulated telomere-length control (Loayza and De Lange, 2003), indicating that the DNA-binding capability of POT1 is vital as a negative regulator of telomere length. The impact of human POT1 on telomere length is complex, since both depletion and overexpression of POT1 lead to telomere lengthening (Ye et al., 2004, Veldman et al., 2004, Colgin et al., 2003, Armbruster et al., 2004). POT1 function as a positive or negative regulator of telomerase activity at the telomere depends on its position of binding relative to the DNA 3′ end and is also modulated by its binding partner, TPP1 (Zaug et al., 2005, Wang et al., 2007, Lei et al., 2005, Kelleher et al., 2005).

Telomerase action at the telomere is highly regulated; it preferentially elongates the shortest telomeres, and recruitment of the enzyme complex to the telomere occurs in mid-S phase of the cell cycle (Bianchi and Shore, 2007, Britt-Compton et al., 2009,Teixeira et al., 2004, Hemann et al., 2001, Tomlinson et al., 2006, Jády et al., 2006). In both budding and fission yeasts, the preference of telomerase to extend the shortest telomeres requires the activity of Tel1, the yeast homolog of human ATM (Sabourin et al., 2007, Hector et al., 2007, Arnerić and Lingner, 2007). ATM and ATR are kinases within the phosphatidylinositol-3 kinase-related kinase (PIKK) family, which regulates cellular responses to DNA damage, mRNA decay, and nutrient-dependent signaling (Lovejoy and Cortez, 2009). Activation of these DNA damage pathways is dampened at telomeres; in mammalian cells, TRF2 represses activation of ATM while POT1 represses ATR (Karlseder et al., 2004, Celli and de Lange, 2005, Denchi and de Lange, 2007, Guo et al., 2007, Okamoto et al., 2013). Nevertheless, there is a large amount of evidence that their yeast homologs play a positive role in facilitating telomere extension by telomerase (Moser et al., 2009, Moser et al., 2011, Yamazaki et al., 2012, Churikov et al., 2013).

It is not known whether the role of the ATM and ATR pathways in recruiting telomerase is conserved in mammals. Although ATM deficiency or ATR mutations can induce telomere shortening or instability in human and mouse cells (Metcalfe et al., 1996, Smilenov et al., 1997, Wong et al., 2003, Wu et al., 2007, Pennarun et al., 2010), these kinases were reported to be dispensable for elongation of the shortest telomeres in mouse models (Feldser et al., 2006, McNees et al., 2010). Also, immortalized cell lines from human patients with ATM mutations are able to maintain their telomeres with telomerase, albeit at short lengths (Sprung et al., 1997). Nonetheless, there is evidence that TRF1-mediated telomere-length regulation in human cells involves ATM. Inhibition of human ATM resulted in increased TRF1 at the telomere, and phosphorylation of TRF1 on serine 367, an ATM/ATR target site, reduced the interaction of TRF1 with telomeres and abrogated its ability to inhibit telomere lengthening (McKerlie et al., 2012, Wu et al., 2007).

In this study, we report that both ATM and ATR are required for the recruitment of human telomerase to telomeres.

ATM and ATR Are Both Required for the Presence of Human Telomerase at Telomeres

Figure 1

ATM and ATR Are Both Required for the Presence of Human Telomerase at Telomeres

(A) Representative images of hTR/telomere FISH in 293T cells treated with the indicated siRNAs or kinase inhibitors. Cells were synchronized to mid-S phase of the cell cycle and probed with hTR probes (green) or a telomere probe (red). Co-localizations are indicated by white arrows in the merge row. Scale bar, 10 μm.

(B) Immunoblot of 293T cells with either ATM (left panel) or ATR (right panel) siRNA-mediated knockdown, using the respective antibodies, with vinculin as a control.

(C) Average co-localizations between telomerase and telomeres in unsynchronized 293T cells treated with control siRNA (gray), ATM siRNA (red) (^∗p = 0.012), or ATR siRNA (purple) (^∗∗p = 0.0095).

(D) Quantitation of decrease in telomerase recruitment in S phase synchronized 293T cells following treatment with two independent ATM and ATR siRNAs; ^∗∗p < 0.01. Cells were synchronized with a thymidine/aphidicolin block (ATM) or sorted into cell-cycle phases by FACS based on DNA content (ATR).

(E) Average telomerase co-localization with telomeres in S phase synchronized 293T cells after treatment with DMSO vehicle (gray), 1.5 μM KU-55933 (light yellow), or 500 nM VE-822 (dark yellow); ^∗p < 0.05.

(F) Telomerase co-localization with telomeres in 293T cells at the indicated number of hours after release from a thymidine/aphidicolin block, treated with control (gray) or ATM (red) siRNA.

(G) Telomerase co-localization with telomeres in 293T cells treated with control (gray) or ATM (red) siRNA. Cells were stained with the DNA dye VyBrant DyeCycle Violet and isolated into cell-cycle phases with FACS. Enrichment of cells in the indicated phases was confirmed by flow cytometry of sorted cells (Figure S1A).

(H) Telomerase co-localization with telomeres in 293T cells treated with control (gray) or ATR (purple) siRNA and sorted by FACS as in Figure 1G.

In all panels, data are presented as the mean of three independent experiments ± SD.

ATM Effect on Telomerase Recruitment Is Partially Mediated by TRF1

Figure 2

ATM Effect on Telomerase Recruitment Is Partially Mediated by TRF1

(A) Immunoblot of 293T cells treated with control or two different TRF1 siRNAs. All lanes are from the same immunoblot, which was cropped to remove intervening irrelevant lanes. Actin was probed as a control.

(B) Average recruitment of telomerase to telomeres in asynchronous 293T cells treated with control (gray) or TRF1 (blue) siRNA;^∗∗p = 0.0011.

(C) Average co-localization of telomerase with telomeres in 293T cells synchronized to G2/M of the cell cycle by release from a thymidine/aphidicolin block; cells were treated with control (gray) or two different TRF1 siRNAs (blue); ^∗p < 0.05.

(D) Telomerase co-localization with telomeres in thymidine/aphidicolin synchronized 293T cells, treated with control (gray) or TRF1 (blue) siRNA. The values along the x axis represent the number of hours since release of cells from G1/S boundary. The control data are the same as those in Figure 1F, since these experiments were performed simultaneously.

(E) Telomerase co-localization with telomeres in 293T cells, treated with control (gray) or TRF1 (blue) siRNA; cells were stained with the DNA dye VyBrant DyeCycle Violet and isolated into the cell-cycle phases with FACS. The control data are the same as those in Figure 1G, since these experiments were performed simultaneously.

(F) FISH for hTR (green) and telomeres (red) in 293T cells treated with control or combined ATM and TRF1 siRNAs. Cells were either asynchronous or synchronized with thymidine and aphidicolin and harvested 3–4 hr (S phase) or 7 hr (G2/M) after release from the G1/S boundary. Co-localizations are indicated by the white arrows in the merge row. Scale bar, 10 μm.

(G) Quantitation of (F); average telomerase-telomere co-localizations after control (gray), ATM only (red), TRF1 only (blue), and ATM/TRF1 (green) siRNAs, in asynchronous, S phase, or G2/M phase cell populations; n = 4; ^∗∗p < 0.01.

Data are presented as the mean of three independent experiments except where indicated otherwise, ± SD.

Phosphorylation of TRF1 at Serine 367 Controls TRF1 and Telomerase Localization to Telomeres

Figure 3

Phosphorylation of TRF1 at Serine 367 Regulates TRF1 and Telomerase Localization to Telomeres

(A) Immunoblot of overexpressed myc-tagged WT TRF1, empty vector, myc-S367A TRF1, or myc-S367D TRF1, probed with an anti-TRF1 antibody and with vinculin probed as a control.

(B) Immunofluorescence with an anti-myc antibody (purple) and hTR/telomere FISH (green and red respectively) in asynchronous 293T cells overexpressing myc-tagged WT, S367A, or S367D TRF1. Co-localizations between hTR and telomeres are indicated by white arrows. Scale bar, 10 μm.

(C) Immunofluorescence with an anti-TRF1 antibody (red) and telomere FISH (green) in 293T cells transfected with empty vector and sorted into S or G2/M phase based on DNA content. Scale bar, 10 μm.

(D) Quantitation of average co-localization of overexpressed myc-tagged TRF1 with telomeres across cell-cycle stages in FACS sorted 293T cells; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005.

(E) Quantitation of total TRF1 at telomeres in S and G2/M phase sorted 293T cells overexpressing myc-tagged WT TRF1, empty vector, or myc-S367D TRF1; ^∗p < 0.05.

(F) Quantitation of average co-localization of hTR and telomeres across cell-cycle stages in FACS-sorted 293T cells overexpressing myc-tagged WT TRF1, myc-S367A TRF1, or myc-S367D TRF1; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005.

In all panels, data are presented as the mean of three independent experiments ± SD.

Stalled Replication Forks Trigger Telomerase Recruitment

Figure 4

Stalled Replication Forks Trigger Telomerase Recruitment

(A) FISH for hTR (green) and telomeres (red) in asynchronous 293T cells treated with control (three left panels) or ATR (three right panels) siRNA. DNA damage was induced by either 30 min of aphidicolin treatment to induce stalled replication forks or 2 Gy gamma irradiation to induce double-strand breaks. Co-localizations between hTR and telomeres are indicated by the white arrows. Scale bar, 10 μm.

(B) Quantitation of telomerase localization to telomeres in cells from Figure 4A; ^∗p < 0.05.

(C) Immunoblot of cells treated as in Figure 4A, probed for ATR, pS1981 ATM, ATM, pT68 Chk2, Chk2, pS345 Chk1, Chk1, and vinculin as a control.

(D) Quantitation of ATM activation (pS1981 ATM levels) from blot in Figure 4C; ^∗p = 0.028.

In all panels, data are presented as the mean of three independent experiments ± SD.

Figure 5

Telomere Elongation Triggered by POT1 Mutation Is Dependent on ATM

(A) Terminal restriction fragment (TRF) assay to determine telomere length. HeLa204 cells were infected with retrovirus encoding luciferase shRNA (ctrl1), no shRNA (ctrl 2), or three different ATM shRNAs, together with myc-POT1ΔOB overexpression, and cultured for 32–37 population doublings (PDs). All lanes are from the same blot, which was cropped to remove intervening irrelevant lanes.

(B) Immunoblot to determine ATM knockdown following shRNA treatment of cells from (A), with α-tubulin as a control.

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Figure 6

ATM Affects Telomerase Assembly

(A) Dot blot for hTR in 293T cells treated with control, ATM, or TRF1 siRNA and synchronized with thymidine/aphidicolin. Crude cell lysates (top panel) contain total levels of cellular hTR, while hTERT immunoprecipitated samples (IP) contain only hTR that has assembled with hTERT. Top row: in-vitro-transcribed hTR standard.

(B) Quantitation of total hTR levels following control (gray), ATM (red), or TRF1 (blue) siRNA treatments (fmol of hTR in 1 × 10⁶cells) from (A); ^∗p = 0.032.

(C) Quantitation of hTR assembled with hTERT (fmol of hTR in 2 × 10⁶ cells) from (A), following control (gray), ATM (red), or TRF1 (blue) siRNA treatments; ^∗p < 0.05, ^∗∗∗p < 0.005.

(D) Quantitation of hTR assembled with hTERT after treatment with combined ATM and TRF1 siRNAs (green); ^∗p < 0.05, ^∗∗∗p < 0.005.

(E) Direct telomerase activity assay of immunoprecipitated telomerase from cells treated with control, ATM, and TRF1 siRNA, synchronized to the indicated cell-cycle stages. LC represents an 18-nt loading control.

(F) Telomerase specific activity, derived from total telomerase activity (Figure 6E) normalized to levels of hTR after hTERT immunoprecipitation (Figure 6C).

(G) Dot blot for hTR in 293T cells treated with control or two different ATR siRNAs and synchronized with thymidine/aphidicolin. The combination of ATR siRNA and thymidine/aphidicolin treatment perturbs progression of the cells through S phase, so this experiment could only be performed on unsynchronized or G1 cells. Crude cell lysates (top panel) contain total levels of cellular hTR, while hTERT immunoprecipitated samples (IP) contain only hTR that has assembled with hTERT. Top row: in-vitro-transcribed hTR standard.

(H) Quantitation of hTR assembled with hTERT after treatment with two different ATR siRNAs (purple); ^∗p < 0.05.

(I) Telomerase specific activity, derived from total telomerase activity (Figure S5K) normalized to levels of hTR after hTERT immunoprecipitation (Figure 6H).

In all panels, data are presented as the mean of three independent experiments ± SD.

Robin Williams death

Posted in Behavior, Biological Networks, Cerebrovascular and Neurodegenerative Diseases, Clinical Diagnostics, Cognition, Curation, Empathy, Gene Regulation, Neurodegenerative Diseases, Neurological Diseases, Neuroscience, tagged clinically different than AD, Cognition, Lewy Bodies, physical deterioration on November 20, 2015| Leave a Comment »

Robin Williams death

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Lewy body: The ‘monster’ dementia blamed for Robin Williams’s death

Robin Williams , wife Susan Schneider and their daughter Zelda Williams at the premiere of "Happy Feet Two" in, November 2011.

Actor Robin Williams with wife Susan Schneider Williams in August 2009. Robin Williams had been planning to undergo neurological testing the week before he committed suicide last year, and likely only had three years to live his widow said earlier this month. Robin Williams , wife Susan Schneider and their daughter Zelda Williams at the premiere of “Happy Feet Two” in, November 2011.

Robin Williams, then 63, had been seeking treatment for severe depression. But last week, his widow, Susan Schneider, revealed that her husband was suffering not only from Parkinson’s disease that had been diagnosed three months earlier, but also from Lewy body dementia (LBD), a progressively brutal illness that affects memory, movement and motor skills, and causes severe psychiatric symptoms.

Schneider says depression didn’t cause Williams’s death: “Lewy body dementia killed Robin. That’s what took his life.”

Strikingly, LBD – sometimes referred to as dementia with Lewy bodies or Parkinson’s with Lewy bodies, depending on symptoms – is the second-most common dementia after Alzheimer’s and affects more than 127,000 Britons. Yet most people have never heard of it.

Robin Williams who suffered from Lewy Body Dementia.

James Galvin, a neurology and psychiatry professor at Florida Atlantic University, says: “It’s the most common disease you’ve never heard of.”

“This disease is a sea monster with 50 tentacles of symptoms that show when they want,” Schneider said.

Williams suffered hallucinations, anxiety, depression, loss of motor control and problem-solving skills, sleep, balance and spatial awareness problems, and delusions.

Schneider describes one incident just weeks before Williams’s death, when she was in the shower and he was standing by the sink.

“Something didn’t seem right,” she recalls, so Schneider got out of the shower to find her husband’s head covered in blood. “He pointed to the door and I said, ‘Did you hit your head?’ and he nodded.” The incident confused her at the time. “But now, finding out all about Lewy body disease, lo and behold, their vision is affected, as is the ability to recognise and identify objects,” she says. “Now I get it.”

Lewy bodies are protein deposits in the brain, explains Professor David Burn, consultant neurologist and director of the biomedical research unit in LBD at the UK National Institute of Health Research (NIHR).

Discovered by Dr Frederic Lewy, a colleague of Dr Alzheimer’s, in 1912, the deposits develop inside nerve cells (neurons) in the brain, interrupting messaging and causing neurons to die. A patient’s symptoms will depend on which part of the brain is affected.

“When neurons die in the cortex, it causes dementia, but when it occurs in the brainstem, it causes motor symptoms (Parkinsonism),” says Burn.

“LBD patients face a rapid deterioration in their cognitive, physical and psychiatric function, and it tends to progress faster than other dementias,” he says.

When Paul Moynagh’s wife, Imogen, couldn’t find her way from a cafe table to the counter on a visit to a National Trust house in Devon in 2006, he thought little of it.Paul, 78, couldn’t have predicted the confusing set of symptoms that Imogen, 74, would experience. LBD is often misdiagnosed as Parkinson’s or Alzheimer’s, and it took doctors almost seven years to confirm her illness.

“Looking back, it began with little signs – loss of spatial awareness is an early symptom – but they were so inconsistent,” recalls Paul, a retired surgeon.

First, there was a minor trembling in Imogen’s hands, then severe sleepiness during the day, along with spasms that made her right foot turn in when she walked. Then she developed depression and suffered panic attacks.

“Imogen has a pragmatic personality,” says Paul. “She used to play sports, was a keen gardener, walked everywhere and looked after our two children, Mark and Rachel. Ten years ago, if you had told her she would be afraid of being left alone, she would have laughed.”

By 2010, Imogen’s reasoning and planning skills were suffering – a key sign of LBD.

A keen bridge player, Imogen recalls: “I stopped winning, so I knew something was wrong.” (Though her speech is now slow, her sense of humour remains.)

Like Schneider, Paul Moynagh was also baffled by his wife’s repeated falls in the years preceding her diagnosis. “She’d had nine different broken bones, breaking her wrist twice, her ankle, and once, when she’d fallen down some stairs, her elbow.”

Imogen, like Williams and many LBD sufferers, was initially diagnosed with Parkinson’s disease.

“She began shuffling when she walked and her voice became weak, both symptoms of Parkinson’s,” says Paul. Meanwhile, her depression was getting worse, not least because Imogen was so aware of what was happening to her.

“It’s different from Alzheimer’s in that people know exactly what’s happening, and one day can be completely lucid and the next be experiencing terrible anxiety and delusions. The more Imogen is aware of her situation, the more she gets depressed.”

When Paul Moynagh’s wife began experiencing hallucinations – a tell-tale sign of LBD – he knew that there was more to her illness than Parkinson’s. “She would see people in the windows of the conservatory and in our floor – which we made look like natural stone – she saw figures speaking to her.

“In my desperation, I would spend hours Googling Imogen’s symptoms until I stumbled on Lewy body dementia,” he says.

Brown says Robert had paranoia and hallucinations – he was frightened by faces he would see in the windows of a summer house he had built at the bottom of the garden. “One evening we were watching the Baftas on television and the camera panned, settling on various stars, and Bob turned to me and said: ‘I think Judi saw us.’ He meant Dame Judi Dench. He thought we were there and became very distressed because he wasn’t correctly dressed.”

“I’m a doctor and I had never even heard of it, and the neurologist was reluctant to accept it, but Imogen ticked all the boxes.” By 2013, a locum psychiatrist finally diagnosed Imogen with LBD. “I went along with my Google list, and she finally made the diagnosis. Two months later, the neurologist finally agreed.”

June Brown, who plays Dot Cotton in EastEnders, lost her husband, actor Robert Arnold, to LBD in 2003.

In a moving video made for the charity Lewy Body Society, Brown recalls: “Bob knew what was happening to him and he hated it. He once said: ‘I never thought I would go like this.'”

Unlike Alzheimer’s sufferers, LBD patients often have lucid memories. “Bob never lost his memory for people’s names. It’s the most strange disease because he would have moments of confusion and moments of clarity. It’s worse than Alzheimer’s because of this awareness of what you’re going through.”

Now, the only way to know that someone had Lewy body dementia is when a post-mortem examination finds Lewy bodies in the brain.

According to LBD specialist Ian McKeith, professor of old age psychiatry at the Newcastle University Institute for Ageing, LBD often gets misdiagnosed because doctors don’t know which questions to ask. He is in the middle of a study funded by the NIHR to develop a diagnostic toolkit for use in NHS practices.

Although there is no cure for LBD, doctors can treat symptoms using drugs that work on the brain’s messaging system, says McKeith. But correct diagnosis is essential. “If antipsychotic or anti-Parkinson’s drugs are given to patients with LBD, they can be fatal,” he says.

“We were living a nightmare,” Susan Schneider said of Robin Williams’s final months.

McKeith says one study found that when carers looking after someone with LBD were asked to rate their quality of life on a scale of zero to one (where zero was as bad as it could be), one in four rated it as below zero.

Still, Paul Moynagh refuses to refer to life with Imogen as a nightmare. She now needs 24-hour attention and help feeding. They recently celebrated their 50th wedding anniversary. During our interview, she turns to her husband and says slowly, with the difficulty she now has in getting words out: “Without your care, I don’t know where I would be.”

“Underneath it all, she is still the lovely person that I married,” he says.

“We still love each other as much as we did before – that hasn’t changed. If anything, I love her more.”

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Biochemistry and Dysmetabolism of Aging and Serious Illness

Posted in Alzheimer's Disease, Amino acids, Biochemical pathways, Biological Networks, Gene Regulation and Evolution, CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiomyopathy, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Cognition, Computational Biology/Systems and Bioinformatics, Curation, DNA repair, Fatty acids, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Genome Biology, Hematology, Hematopoiesis, Human aging, Kinase, Lipid metabolism, Metabolism, Neurodegenerative Diseases, Oxidative phosphorylation, Proteins, Proteomics, Schizophrenia, Signaling, Signaling & Cell Circuits, Warburg effect, tagged Cancer - General, IGF1, Insulin, mitochondria, Oxidative phosphorylation, skeletal and cardiomyocyte on November 19, 2015| Leave a Comment »

Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002

Highlights

•Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
•Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
•Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A₂ sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H₂O₂ production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H₂O₂ production to activation of the cPLA₂-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H₂O₂ activation of cPLA₂-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA₂-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H₂O₂, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA₂-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

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Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H₂O₂ (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H₂O₂ functions as a signaling molecule to activate cPLA₂, the initiating enzyme in the cPLA₂-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA₂ immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA₂-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA₂ (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H₂O₂ production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA₂ by physiologically relevant concentrations of H₂O₂ was confirmed in vitro. Astrocytic involvement in the cPLA₂-sphingomyelinase pathway was also indicated by an increase in cPLA₂ positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA₂immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA₂, neuronal cPLA₂ is activated by an influx of intracellular calcium, whereas astrocytic cPLA₂ is directly activated by excessive generation of H₂O₂ (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA₂ in astrocytes by H₂O₂ and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA₂ is the cellular mediator of the H₂O₂ dependent cPLA₂-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H₂O₂ in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA₂, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA₂-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H₂O₂generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9. http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.

Ricciardi MR¹, Mirabilii S², Allegretti M², Licchetta R², Calarco A³, Torrisi MR¹, Foà R², Nicolai R⁴, Peluso G³, Tafuri A¹. Author information

Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.

Oncogene. 2015 Oct 12. http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]

Tumour-suppression function of KLF12 through regulation of anoikis.

Godin-Heymann N¹, Brabetz S¹, Murillo MM^1,², Saponaro M³, Santos CR⁴, Lobley A⁵, East P⁵, Chakravarty P⁵, Matthews N⁶, Kelly G⁵, Jordan S¹, Castellano E¹, Downward J^1,². Author information

Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.

Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.

Montal ED¹, Dewi R², Bhalla K³, Ou L², Hwang BJ², Ropell AE⁴, Gordon C⁴, Liu WJ³, DeBerardinis RJ⁵, Sudderth J⁵, Twaddel W⁶, Boros LG⁷, Shroyer KR⁴,Duraisamy S⁸, Drapkin R⁸, Powers RS⁶, Rohde JM⁹, Boxer MB⁹, Wong KK¹⁰, Girnun GD¹¹. Author information

Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.

Mol Cancer Res. 2015 Oct;13(10):1408-20. http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.

Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.

Kardos GR¹, Wastyk HC², Robertson GP³. Author information

Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway. This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.

J Natl Cancer Inst. 2015 Oct 12;108(1). pii: djv287. http://dx.doi.org:/10.1093/jnci/djv287.

SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.

Saxena N¹, Maio N¹, Crooks DR¹, Ricketts CJ¹, Yang Y¹, Wei MH¹, Fan TW¹, Lane AN¹, Sourbier C¹, Singh A¹, Killian JK¹, Meltzer PS¹, Vocke CD¹, Rouault TA¹, Linehan WM¹. Author information

BACKGROUND: Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.

METHODS: In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

Cancer Res. 2015 Oct 15;75(20):4429-36. http://dx.doi.org:/10.1158/0008-5472.CAN-15-0074.

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Feeley KP¹, Bray AW¹, Westbrook DG¹, Johnson LW², Kesterson RA², Ballinger SW¹, Welch DR³. Author information

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4. http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]

Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Ju R¹, Guo L¹, Li J¹, Zhu L¹, Yu X¹, Chen C¹, Chen W¹, Ye C², Zhang D³. Author information

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

Cancer Immunol Res. 2015 Nov;3(11):1236-47. http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Hossain F¹, Al-Khami AA², Wyczechowska D², Hernandez C², Zheng L², Reiss K³, Valle LD⁴, Trillo-Tinoco J², Maj T⁵, Zou W⁵, Rodriguez PC⁶, Ochoa AC⁷. Author information

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0. http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

Sato PY¹, Chuprun JK¹, Ibetti J¹, Cannavo A¹, Drosatos K¹, Elrod JW¹, Koch WJ². Author information

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]

All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.

Yang L¹, Luo C², Chen C¹, Wang X¹, Shi W¹, Liu J¹. Author information

BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

J Biol Chem. 2015 Oct 23;290(43):25834-46. http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.

Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

Trefely S¹, Khoo PS², Krycer JR³, Chaudhuri R³, Fazakerley DJ³, Parker BL³, Sultani G¹, Lee J⁴, Stephan JP⁴, Torres E⁴, Jung K⁴, Kuijl C⁴, James DE⁵,Junutula JR⁶, Stöckli J⁷. Author information

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

FASEB J. 2015 Oct 19. http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]

Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Cho Y¹, Hazen BC¹, Gandra PG¹, Ward SR¹, Schenk S¹, Russell AP¹, Kralli A². Author information

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Cell Death Dis. 2015 Oct 29;6:e1944. http://dx.doi.org:/10.1038/cddis.2015.306.

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

Mughal W^1,², Nguyen L², Pustylnik S³, da Silva Rosa SC^1,², Piotrowski S^1,², Chapman D^2,⁴, Du M³, Alli NS³, Grigull J^3,⁵, Halayko AJ^6,⁷, Aliani M⁸, Topham MK⁹, Epand RM¹⁰, Hatch GM^2,¹¹, Pereira TJ^2,¹¹, Kereliuk S^2,¹¹, McDermott JC³, Rampitsch C¹², Dolinsky VW^2,¹¹, Gordon JW^1,^2,⁴. Author information

Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

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Gene-Silencing and Gene-Disabling in Pharmaceutical Development

Posted in CRISPR/Cas9 & Gene Editing, Curation, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gene Regulation, Genetics & Innovations in Treatment, RNA Biology, RNA Biology, Cancer and Therapeutics, tagged CRISPR/Cas, Drug discovery, RNAi, therapeutics on November 19, 2015| Leave a Comment »

Gene-Silencing and Gene-Disabling in Pharmaceutical Development

Larry H. Bernstein, MD, FCAP, Curator

LPBI

2.1.2.8

Gene-Silencing and Gene-Disabling in Pharmaceutical Development, 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

Down and Out with RNAi and CRISPR

http://www.genengnews.com/gen-articles/down-and-out-with-rnai-and-crispr/5619/

RNA interference (RNAi) silences, or knocks down, the translation of a gene by inducing degradation of a gene target’s transcript. To advance RNAi applications, Thermo Fisher Scientific has developed two types of small RNA molecules: short interfering RNAs and microRNAs. The company also offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications.

Genes can be knocked down with RNA interference (RNAi) or knocked out with CRISPR-Cas9. RNAi, the screening workhorse, knocks down the translation of genes by inducing rapid degradation of a gene target’s transcript.

CRISPR-Cas9, the new but already celebrated genome-editing technology, cleaves specific DNA sequences to render genes inoperative. Although mechanistically different, the two techniques complement one another, and when used together facilitate discovery and validation of scientific findings.

RNAi technologies along with other developments in functional genomics screening were discussed by industry leaders at the recent Discovery on Target conference. The conference, which emphasized the identification and validation of novel drug targets and the exploration of unknown cellular pathways, included a symposium on the development of CRISPR-based therapies.

RNAi screening can be performed in either pooled or arrayed formats. Pooled screening provides an affordable benchtop option, but requires back-end deconvolution and deep sequencing to identify the shRNA causing the specific phenotype. Targets are much easier to identify using the arrayed format since each shRNA clone is in an individual well.

“CRISPR complements RNAi screens,” commented Ryan Raver, Ph.D., global product manager of functional genomics at Sigma-Aldrich. “You can do a whole genome screen with either small interfering RNA (siRNA) or small hairpin RNA (shRNA), then validate with individual CRISPRs to ensure it is a true result.”

A powerful and useful validation method for knockdown or knockout studies is to use lentiviral open reading frames (ORFs) for gene re-expression, for rescue experiments, or to detect whether the wild-type phenotype is restored. However, the ORF randomly integrates into the genome. Also, with this validation technique, gene expression is not acting under the endogenous promoter. Accordingly, physiologically relevant levels of the gene may not be expressed unless controlled for via an inducible system.

In the future, CRISPR activators may provide more efficient ways to express not only wild-type but also mutant forms of genes under the endogenous promoter.

Choice of screening technique depends on the researcher and the research question. Whole gene knockout may be necessary to observe a phenotype, while partial knockdown might be required to investigate functions of essential or lethal genes. Use of both techniques is recommended to identify all potential targets.

For example, recently, a whole genome siRNA screen on a human glioblastoma cell line identified a gene, known as FAT1, as a negative regulator of apoptosis. A CRISPR-mediated knockout of the gene also conferred sensitivity to death receptor–induced apoptosis with an even stronger phenotype, thereby validating FAT1’s new role and link to extrinsic apoptosis, a new model system.

Dr. Raver indicated that next-generation RNAi libraries that are microRNA-adapted might have a more robust knockdown of gene expression, up to 90–95% in some cases. Ultracomplex shRNA libraries help to minimize both false-negative and false-positive rates by targeting each gene with ~25 independent shRNAs and by including thousands of negative-control shRNAs.

Recently, a relevant paper emerged from the laboratory of Jonathan Weissman, Ph.D., a professor of cellular and molecular pharmacology at the University of California, San Francisco. The paper described how a new ultracomplex pooled shRNA library was optimized by means of a microRNA-adapted system. This system, which was able to achieve high specificity in the detection of hit genes, produced robust results. In fact, they were comparable to results obtained with a CRISPR pooled screen. Members of the Weissman group systematically optimized the promoter and microRNA contexts for shRNA expression along with a selection of guide strands.

Using a sublibrary of proteostasis genes (targeting 2,933 genes), the investigators compared CRISPR and RNAi pooled screens. Data showed 48 hits unique to RNAi, 40 unique to CRISPR, and an overlap of 21 hits (with a 5% false discovery rate cut-off). Together, the technologies provided a more complete research story.

“RNA screens are well accepted and will continue to be used, but it is important biologically that researchers step away from the RNA mechanism to further study and validate their hits to eliminate potential bias,” explained Louise Baskin, senior product manager, Dharmacon, part of GE Healthcare. “The natural progression is to adopt CRISPR in the later stages.”

RNAi uses the cell’s endogenous mechanism. All of the components required for gene knockdown are already within the cell, and the delivery of the siRNA starts the process. With the CRISPR gene-editing system, which is derived from a bacterial immune defense system, delivery of both the guide RNA and the Cas9 nuclease, often the rate limiter in terms of knockout efficiency, are required.

Arrayed CRISPR Screens

Synthetic crRNA:tracrRNA reagents can be used in a similar manner to siRNA reagents for assessment of phenotypes in a cell population. Top row: A reporter cell line stably expressing Cas9 nuclease was transfected with GE Dharmacon’s Edit-R synthetic crRNA:tracrRNA system, which was used to target three positive control genes (PSMD7, PSMD14, and VCP) and a negative control gene (PPIB). Green cells indicate EGFP signaling occuring as a result of proteasome pathway disruption. Bottom row: A siGENOME siRNA pool targeting the same genes was used in the same reporter cell line.

In pooled approaches, the cell has to either drop out or overexpress so that it is sortable, limiting the types of addressable biological questions. A CRISPR-arrayed approach opens up the door for use of other analytical tools, such as high-content imaging, to identify hits of interest.

To facilitate use of CRISPR, GE recently introduced Dharmacon Edit-R synthetic CRISPR RNA (crRNA) libraries that can be used to carry out high-throughput arrayed analysis of multiple genes. Rather than a vector- or plasmid-based gRNA to guide the targeting of the Cas9 cleavage, a synthetic crRNA and tracrRNA are used. These algorithm-designed crRNA reagents can be delivered into the cells very much like siRNA, opening up the capability to screen multiple target regions for many different genes simultaneously.

GE showed a very strong overlap between CRISPR and RNAi using this arrayed approach to validate RNAi screen hits with synthetic crRNA. The data concluded that CRISPR can be used for medium- or high-throughput validation of knockdown studies.

“We will continue to see a lot of cooperation between RNAi and gene editing,” declared Baskin. “Using the CRISPR mechanism to knockin could introduce mutations to help understand gene function at a much deeper level, including a more thorough functional analysis of noncoding genes.

“These regulatory RNAs often act in the nucleus to control translation and transcription, so to knockdown these genes with RNAi would require export to the cytoplasm. Precision gene editing, which takes place in the nucleus, will help us understand the noncoding transcriptome and dive deeper into how those genes regulate disease progression, cellular development and other aspects of human health and biology.”

Tool Selection

The functional genomics tool should fit the specific biology; the biology should not be forced to fit the tool. Points to consider include the regulation of expression, the cell line or model system, as well as assay scale and design. For example, there may be a need for regulatable expression. There may be limitations around the cell line or model system. And assay scale and design may include delivery conditions and timing to optimally complete perturbation and reporting.

“Both RNAi- and CRISPR-based gene modulation strategies have pros and cons that should be considered based on the biology of the genes being studied,” commented Gwen Fewell, Ph.D., chief commercial officer, Transomic Technologies.

RNAi reagents, which can produce hypomorphic or transient gene-suppression states, are well known for their use in probing drug targets. In addition, these reagents are enriching studies of gene function. CRISPR-Cas9 reagents, which produce clean heterozygous and null mutations, are important for studying tumor suppressors and other genes where complete loss of function is desired.

Schematic of a pooled shRNA screening workflow developed by Transomic Technologies. Cells are transduced, and positive or negative selection screens are performed. PCR amplification and sequencing of the shRNA integrated into the target cell genome allows the determination of shRNA representation in the population.

Timing to readout the effects of gene perturbation must be considered to ensure that the biological assay is feasible. RNAi gene knockdown effects can be seen in as little as 24–72 hours, and inducible and reversible gene knockdown can be realized. CRISPR-based gene knockout effects may become complete and permanent only after 10 days.

Both RNAi and CRISPR reagents work well for pooled positive selection screens; however, for negative selection screens, RNAi is the more mature tool. Current versions of CRISPR pooled reagents can produce mixed populations containing a fraction of non-null mutations, which can reduce the overall accuracy of the readout.

To meet the needs of varied and complex biological questions, Transomic Technologies has developed both RNAi and CRISPR tools with options for optimal expression, selection, and assay scale. For example, the company’s shERWOOD-UltramiR shRNA reagents incorporate advances in design and small RNA processing to produce increased potency and specificity of knockdown, particularly important for pooled screens.

Sequence-verified pooled shRNA screening libraries provide flexibility in promoter choice, in vitro formats, in vivo formats, and a choice of viral vectors for optimal delivery and expression in biologically relevant cell lines, primary cells or in vivo.

The company’s line of lentiviral-based CRISPR-Cas9 reagents has variable selectable markers such that guide RNA- and Cas9-expressing vectors, including inducible Cas9, can be co-delivered and selected for in the same cell to increase editing efficiency. Promoter options are available to ensure expression across a range of cell types.

“Researchers are using RNAi and CRISPR reagents individually and in combination as cross-validation tools, or to engineer CRISPR-based models to perform RNAi-based assays,” informs Dr. Fewell. “Most exciting are parallel CRISPR and RNAi screens that have tremendous potential to uncover novel biology.”

Converging Technologies

The convergence of RNAi technology with genome-editing tools, such as CRISPR-Cas9 and transcription activator-like effector nucleases, combined with next-generation sequencing will allow researchers to dissect biological systems in a way not previously possible.

“From a purely technical standpoint, the challenges for traditional RNAi screens come down to efficient delivery of the RNAi reagents and having those reagents provide significant, consistent, and lasting knockdown of the target mRNAs,” states Ross Whittaker, Ph.D., a product manager for genome editing products at Thermo Fisher Scientific. “We have approached these challenges with a series of reagents and siRNA libraries designed to increase the success of RNAi screens.”

Thermo Fisher’ provides lipid-transfection RNAiMax reagents, which effectively deliver siRNA. In addition, the company’s Silencer and Silencer Select siRNA libraries provide consistent and significant knockdown of the target mRNAs. These siRNA libraries utilize highly stringent bioinformatic designs that ensure accurate and potent targeting for gene-silencing studies. The Silencer Select technology adds a higher level of efficacy and specificity due to chemical modifications with locked nucleic acid (LNA) chemistry.

The libraries alleviate concerns for false-positive or false-negative data. The high potency allows less reagent use; thus, more screens or validations can be conducted per library.

Dr. Whittaker believes that researchers will migrate regularly between RNAi and CRISPR-Cas9 technology in the future. CRISPR-Cas9 will be used to create engineered cell lines not only to validate RNAi hits but also to follow up on the underlying mechanisms. Cell lines engineered with CRISPR-Cas9 will be utilized in RNAi screens. In the long term, CRISPR-Cas9 screening will likely replace RNAi screening in many cases, especially with the introduction of arrayed CRISPR libraries.

Validating Antibodies with RNAi

Unreliable antibody specificity is a widespread problem for researchers, but RNAi is assuaging scientists’ concerns as a validation method.

The procedure introduces short hairpin RNAs (shRNAs) to reduce expression levels of a targeted protein. The associated antibody follows. With its protein knocked down, a truly specific antibody shows dramatically reduced or no signal on a Western blot. Short of knockout animal models, RNAi is arguably the most effective method of validating research antibodies.

The method is not common among antibody suppliers—time and cost being the chief barriers to its adoption, although some companies are beginning to embrace RNAi validation.

“In the interest of fostering better science, Proteintech felt it was necessary to implement this practice,” said Jason Li, Ph.D., founder and CEO of Proteintech Group, which made RNAi standard protocol in February 2015. “When researchers can depend on reproducibility, they execute more thorough experiments and advance the treatment of human diseases and conditions.”

Down and Out with RNAi and CRISPR

RNA-Based Therapeutics and Vaccines

RNA-based biopharmaceuticals, which includes therapeutics and vaccines, is a relatively new class of treatment and prophylactic for a number of chronic and rare diseases, including cancer, diabetes, tuberculosis, and certain cardiovascular conditions. The field holds great promise in the prevention and treatment of these diseases as demonstrated by early-phase clinical trials as well as significant investment by the drug development community.

Ready, Aim, CRISPR (or RNAi)

Recent progress in probing gene function via the RNAi and CRISPR methods were a strong theme of the Discovery On Target conference. Both methods enable researchers to impair the function of a targeted gene.

Masked RNAi Drug Slips through Membrane, Sheds Guise within Cell

For small interfering RNA, approaching a cell is like walking up to the door of an old speakeasy. Such doors were heavily reinforced and had a narrow, built-in sliding panel at eye level, and if the eyes peering out though the open panel didn’t like the look of you, well, you were not getting inside. Failing to gain entry is something that happens all too frequently to small interfering RNAs, which admittedly are anything but “life of the party” types.

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Rheumatoid arthritis update

Posted in Biological Networks, Gene Regulation and Evolution, Biomedical Measurement Science, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Conference Coverage with Social Media, Curation, Cytokines, Disease Biology, Small Molecules in Development of Therapeutic Drugs, FDA Regulatory Affairs, Gene Regulation, Immunology, Innovations, Metabolism, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Population Health Management, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Signaling & Cell Circuits, Systemic Inflammatory Response Related Disorders, Translational Science on November 19, 2015| Leave a Comment »

Rheumatoid arthritis update

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Innovation update: Advancing the standard of care in rheumatoid arthritis

By Nicole Gray | November 19, 2015

http://www.biopharmadive.com/news/innovation-update-advancing-the-standard-of-care-in-rheumatoid-arthritis/409496/

This year’s American College of Rheumatology (ACR) meeting, which took place in San Francisco from November 6 to 11, reflected an unprecedented level of innovation, including numerous advances in the area of rheumatoid arthritis (RA).

BioPharma Dive spoke with executives from Pfizer and Eli Lilly to discuss the new treatment landscape and ongoing innovation in this therapeutic space.

Old innovation makes way for new innovation

Twenty years ago, the standard of care for RA was some combination of basic NSAIDS, along with methotrexate. Caregivers focused on symptom relief, and it was widely understood that many patients would fail to achieve remission. As the disease developed, patients would eventually develop severely life-limiting disabilities as their disease progressed.

During this period, researchers presenting at conferences grew excited about data on a new class of drugs known as anti-tumor necrosis factor (TNF) antibodies. In an article published in Acta Orthopaedica Scandinavica in 1995, two physician-researchers wrote the following:

“Primary results have recently been published on the use of anti-TNF monoclonal antibodies. In a controlled trial these antibodies were able to significantly influence a number of disease-activity variables in RA. An important observation was that the clinical effect lasted from weeks to, in some cases, months. Although the potential of these agents for clinical use is still uncertain, these observations suggest that interfering with certain targets of the immune-inflammatory process is possible, effective and so far without side effects.”

About four years after Drs. Van de Putte and Van Riel extolled the virtues of disease-modifying biologics in clinical trials, the first anti-TNF antibody, Remicade (infliximab) was approved in 1999. At that point, the standard of care for RA improved significantly, forever changing the treatment paradigm for patients with RA.

The expanding class of JAK inhibitors

At this year’s ACR meeting, researchers focused on anti-inflammatory antibodies and a relatively new class of oral drugs known as janus kinase (JAK) inhibitors. Interest in JAK inhibitors has spiked since the approval of Pfizer’s oral medication Xeljanz (tofacitinib) —the first, and currently the only, JAK inhibitor approved for the treatment of moderate-to-severe RA.JAK inhibitors have garnered interest because of the role they can play in expanding a treatment area dominated by synthetic and biologic disease-modifying anti-rheumatic drugs (DMARDs). Could JAK inhibitors provide the breakthrough in RA that the anti-TNF antibodies provided almost 20 years ago?

Currently, Eli Lilly and Incyte are in late-stage development of baricitinib, a JAK1/JAK2 inhibitor for treatment of RA. Until last December, Johnson & Johnson (J&J) and Astellas were working jointly on another JAK inhibitor, known as ASPO15K, but J&J exercised its opt-out option and left the partnership. Astellas vowed to go it alone or look for a new partner, but there have not been many updates on ASPO15K within the last year.

Innovation means understanding and responding to unmet needs

Like many other therapeutic areas, RA treatments are often used in combination. For some patients, the combination of methotrexate and a powerful biologic, such as Remicade (infliximab), will help a patient achieve remission Yet others will either not respond to methotrexate and Remicade, or will have a negative reaction. Understanding how to help nonresponders achieve relief has become a key area of research in RA.

According to Terence Rooney, MD, Medical Director at Lilly Bio-Medicines, “A substantial proportion of patients treated with methotrexate – commonly used across the disease continuum for 25 years – do not achieve satisfactory disease control, signaling a need for more effective RA treatment options. In addition, studies have shown that some patients who initially respond to biologics lose response over time, and approximately 40 percent of patients with high disease activity never respond adequately to TNF antagonist biologics.”

Innovative clinical trial design

As Lilly and Incyte approach the end of the development process for baricitinib, they have been collecting results from clinical trials designed to both establish basic efficacy and safety in placebo-controlled and comparator trials, and to obtain data on targeted patient populations.

According to Rooney, “The baricitinib phase three program investigated the benefit of baricitinib across the spectrum of patients with rheumatoid arthritis, including newly diagnosed patients, patients who had failed to respond to conventional DMARDs, and patients who had failed multiple injectable biologic DMARD therapies.”

“In addition, the phase 3 program included two 52-week studies that incorporated either methotrexate or adalimumab as active comparators to provide useful information for therapeutic positioning of baricitinib. In these studies, baricitinib was statistically superior to methotrexate and to adalimumab in improving signs and symptoms, physical function, and important patient-reported outcomes including pain, fatigue and stiffness.”

Rooney also pointed out that there is additional data establishing baricitinib as a DMARD that significantly inhibits progressive radiographic joint damage.

Experience plus evidence equals more innovation

As has become the norm, companies at ACR often highlight new data confirming the efficacy and safety of already approved drugs in larger patient populations and in real-world settings..

Lilly currently has data on more than 40,000 patients worldwide, reflecting its global ambitions. Assuming that baricitinib is approved next year (the goal is to file at the end of the year), Lilly will continue to present data at ACR in the coming years highlighting the results of its long-term extension study, RA-BEYOND.

Pfizer’s up-to-date Xeljanz data presentation at ACR

Although Xeljanz has been on the market for three years in more than 40 countries, Pfizer continues to focus on collecting new data and using it to expand use of Xeljanz. In fact, Pfizer had 20 abstracts focused solely on Xeljanz at ACR 2015.

According to Rory O’Connor, MD, Senior Vice President and Head of Global Medical Affairs, Global Innovative Pharmaceuticals Business, Pfizer, “Ongoing clinical trials and long-term extension studies provide important information about the safety and efficacy of Xeljanz in RA. We are focused on continuing to build on our knowledge of the clinical application of Xeljanz in real-world settings.”

Pfizer was also able to highlight new data that supports their recent NDA for Xeljanz XR, a once-daily formulation of Xeljanz, which is currently approved as a twice-daily dosing formulation.

JAK inhibition beyond RA

One of the most exciting things about the progress with JAK inhibitors is the possibility to innovate treatments beyond RA. Lilly has been exploring the role of JAK-dependent cytokines in the pathogenesis of numerous inflammatory and autoimmune diseases. The company also plans to meet with regulatory authorities to develop a pediatric program for juvenile RA and idiopathic arthritis.

Meanwhile, Pfizer has developed a broad portfolio of various JAK inhibitors and therapies with new modes of action. Already, Pfizer researchers have completed two phase three studies in ulcerative colitis and the top-line results have been positive.

Medical meetings are exciting, because they provide a forum for discussing breakthroughs and portending a future in which the standard of care improves. For companies like Lilly, Incyte, and Pfizer, continual development of more novel approaches to serious diseasesis like a call-response echo chamber in which innovation drives more innovation, resulting in better long-term outcomes for patients.

The JAK/STAT signaling pathway

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Journal of Cell Science 2004 117: 1281-1283; http://dx.doi.org:/10.1242/jcs.00963

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is one of a handful of pleiotropic cascades used to transduce a multitude of signals for development and homeostasis in animals, from humans to flies. In mammals, the JAK/STAT pathway is the principal signaling mechanism for a wide array of cytokines and growth factors. JAK activation stimulates cell proliferation, differentiation, cell migration and apoptosis. These cellular events are critical to hematopoiesis, immune development, mammary gland development and lactation, adipogenesis, sexually dimorphic growth and other processes. Predictably, mutations that reduce JAK/STAT pathway activity affect these processes (reviewed by Igaz et al., 2001; O’Shea et al., 2002). Conversely, mutations that constitutively activate or fail to regulate JAK signaling properly cause inflammatory disease, erythrocytosis, gigantism and an array of leukemias. Here we present a general overview of the JAK/STAT pathway and illustrate the primary mechanisms of activation and regulation of this essential signaling cascade.

Mechanistically, JAK/STAT signaling is relatively simple, with only a few principal components (for reviews, see Aaronson and Horvath, 2002; Heinrich et al., 2003; Kisseleva et al., 2002; O’Shea et al., 2002). As described above, a variety of ligands and their receptors stimulate the JAK/STAT pathway. Intracellular activation occurs when ligand binding induces the multimerization of receptor subunits. For some ligands, such as erythropoietin and growth hormone, the receptor subunits are bound as homodimers while, for others, such as interferons and interleukins, the receptor subunits are heteromultimers. For signal propagation through either homodimers or heteromultimers, the cytoplasmic domains of two receptor subunits must be associated with JAK tyrosine kinases. JAKs are distinctive in that they have tandem kinase-homologous domains at the C-terminus. The first is a non-catalytic regulatory domain, whereas the second has tyrosine kinase activity. In mammals, the JAK family comprises four members: JAK1, JAK2, JAK 3 and Tyk2. JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity, allowing trans-phosphorylation. The activated JAKs subsequently phosphorylate additional targets, including both the receptors and the major substrates, STATs. STATs are latent transcription factors that reside in the cytoplasm until activated. The seven mammalian STATs bear a conserved tyrosine residue near the C-terminus that is phosphorylated by JAKs. This phosphotyrosine permits the dimerization of STATs through interaction with a conserved SH2 domain. Phosphorylated STATs enter the nucleus by a mechanism that is dependent on importin α-5 (also called nucleoprotein interactor 1) and the Ran nuclear import pathway. Once in the nucleus, dimerized STATs bind specific regulatory sequences to activate or repress transcription of target genes. Thus the JAK/STAT cascade provides a direct mechanism to translate an extracellular signal into a transcriptional response.

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In addition to the principal components of the pathway, other effector proteins have been identified that contribute to at least a subset of JAK/STAT signaling events. STAMs (signal-transducing adapter molecules) are adapter molecules with conserved VHS and SH3 domains (Lohi and Lehto, 2001). STAM1 and STAM2A can be phosphorylated by JAK1-JAK3 in a manner that is dependent on a third domain present in some STAMs, the ITAM (inducible tyrosine-based activation motif). Through a poorly understood mechanism, the STAMs facilitate the transcriptional activation of specific target genes, including MYC. A second adapter that facilitates JAK/STAT pathway activation is StIP (stat-interacting protein), a WD40 protein. StIPs can associate with both JAKs and unphosphorylated STATs, perhaps serving as a scaffold to facilitate the phosphorylation of STATs by JAKs. A third class of adapter with function in JAK/STAT signaling is the SH2B/Lnk/APS family. These proteins contain both pleckstrin homology and SH2 domains and are also substrates for JAK phosphorylation. Both SH2-Bβ and APS associate with JAKs, but the former facilitates JAK/STAT signaling while the latter inhibits it. The degree to which each of these adapter families contributes to JAK/STAT signaling is not yet well understood, but it is clear that various proteins outside the basic pathway machinery influence JAK/STAT signaling.

In addition to JAK/STAT pathway effectors, there are three major classes of negative regulator: SOCS (suppressors of cytokine signaling), PIAS (protein inhibitors of activated stats) and PTPs (protein tyrosine phosphatases) (reviewed by Greenhalgh and Hilton, 2001). Perhaps the simplest are the tyrosine phosphatases, which reverse the activity of the JAKs. The best characterized of these is SHP-1, the product of the mouse motheaten gene. SHP-1 contains two SH2 domains and can bind to either phosphorylated JAKs or phosphorylated receptors to facilitate dephosphorylation of these activated signaling molecules. Other tyrosine phosphatases, such as CD45, appear to have a role in regulating JAK/STAT signaling through a subset of receptors.

SOCS proteins are a family of at least eight members containing an SH2 domain and a SOCS box at the C-terminus (reviewed by Alexander, 2002). In addition, a small kinase inhibitory region located N-terminal to the SH2 domain has been identified for SOCS1 and SOCS3. The SOCS complete a simple negative feedback loop in the JAK/STAT circuitry: activated STATs stimulate transcription of the SOCS genes and the resulting SOCS proteins bind phosphorylated JAKs and their receptors to turn off the pathway. The SOCS can affect their negative regulation by three means. First, by binding phosphotyrosines on the receptors, SOCS physically block the recruitment of signal transducers, such as STATs, to the receptor. Second, SOCS proteins can bind directly to JAKs or to the receptors to specifically inhibit JAK kinase activity. Third, SOCS interact with the elongin BC complex and cullin 2, facilitating the ubiquitination of JAKs and, presumably, the receptors. Ubiquitination of these targets decreases their stability by targeting them for proteasomal degradation.

The third class of negative regulator is the PIAS proteins: PIAS1, PIAS3, PIASx and PIASy. These proteins have a Zn-binding RING-finger domain in the central portion, a well-conserved SAP (SAF-A/Acinus/PIAS) domain at the N-terminus, and a less-well-conserved carboxyl domain. The latter domains are involved in target protein binding. The PIAS proteins bind to activated STAT dimers and prevent them from binding DNA. The mechanism by which PIAS proteins act remains unclear. However, PIAS proteins have recently been demonstrated to associate with the E2 conjugase Ubc9 and to have E3 conjugase activity for sumoylation that is mediated by the RING finger domain (reviewed by Jackson, 2001). Although there is evidence that STATs can be modified by sumoylation (Rogers et al., 2003), the function of that modification in negative regulation is not yet known.

Although the mechanism of JAK/STAT signaling is relatively simple in theory, the biological consequences of pathway activation are complicated by interactions with other signaling pathways (reviewed by Heinrich et al., 2003; Rane and Reddy, 2000; Shuai, 2000). An understanding of this cross-talk is only beginning to emerge, but the best characterized interactions of the JAK/STAT pathway are with the receptor tyrosine kinase (RTK)/Ras/MAPK (mitogen-activated protein kinase) pathway. The relationship between these cascades is complex and their paths cross at multiple levels, each enhancing activation of the other. First, activated JAKs can phosphorylate tyrosines on their associated receptors that can serve as docking sites for SH2-containing adapter proteins from other signaling pathways. These include SHP-2 and Shc, which recruit the GRB2 adapter and stimulate the Ras cascade. The same mechanism stimulates other cascades, such as the recruitment and JAK phosphorylation of insulin receptor substrate (IRS) and p85, which results in the activation of the phosphoinositide 3-kinase (PI3K) pathway [for more on PI3K signaling, see Foster et al. (Foster et al., 2003)]. JAK/STAT signaling also indirectly promotes Ras signaling through the transcriptional activation of SOCS3. SOCS3 binds RasGAP, a negative regulator of Ras signaling, and reduces its activity, thereby promoting activation of the Ras pathway. Reciprocally, RTK pathway activity promotes JAK/STAT signaling by at least two mechanisms. First, the activation of some RTKs, including EGFR and PDGFR, results in the JAK-independent tyrosine phosphorylation of STATs, probably by the Src kinase. Second, RTK/Ras pathway stimulation causes the downstream activation of MAPK. MAPK specifically phosphorylates a serine near the C-terminus of most STATs. While not absolutely necessary for STAT activity, this serine phosphorylation dramatically enhances transcriptional activation by STAT. In addition to RTK and PI3K interactions with JAK/STAT signaling, multiple levels of cross-talk with the TGF-β signaling pathway have been recently reported [for a review of TGF-β, see (Moustakas, 2002)]. Furthermore, the functions of activated STATs can be altered through association with other transcription factors and cofactors that are regulated by other signaling pathways. Thus the integration of input from many signaling pathways must be considered if we are to understand the biological consequences of cytokine stimulation.

References

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Aaronson, D. S. and Horvath, C. M. (2002). A road map for those who know JAK-STAT. Science 296, 1653-1655.

Abstract/FREE Full Text
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Agaisse, H., Petersen, U. M., Boutros, M., Mathey-Prevot, B. and Perrimon, N. (2003). Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev. Cell 5, 441-450.

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Alexander, W. S. (2002). Suppressors of cytokine signalling (SOCS) in the immune system. Nat. Rev. Immunol. 2,410-416.

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Dearolf, C. R. (1999). JAKs and STATs in invertebrate model organisms. Cell. Mol. Life Sci. 55, 1578-1584.

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Foster, F. M., Traer, C. J., Abraham, S. M. and Fry, M. J. (2003). The phosphoinositide (PI) 3-kinase family. J. Cell Sci.116, 3037-3040.

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https://youtu.be/9JHBHSHaBeI

Published on 27 Feb 2014

The JAK/STAT secondary messenger signaliing pathway..
Presented by: Joseph Farahany, M.D

Jak/Stat Signaling Pathway

Jaks and Stats are critical components of many cytokine receptor systems; regulating growth, survival, differentiation, and pathogen resistance. An example of these pathways is shown for the IL-6 (or gp130) family of receptors, which coregulate B cell differentiation, plasmacytogenesis, and the acute phase reaction. Cytokine binding induces receptor dimerization, activating the associated Jaks, which phosphorylate themselves and the receptor. The phosphorylated sites on the receptor and Jaks serve as docking sites for the SH2-containing Stats, such as Stat3, and for SH2-containing proteins and adaptors that link the receptor to MAP kinase, PI3K/Akt, and other cellular pathways.

Phosphorylated Stats dimerize and translocate into the nucleus to regulate target gene transcription. Members of the suppressor of cytokine signaling (SOCS) family dampen receptor signaling via homologous or heterologous feedback regulation. Jaks or Stats can also participate in signaling through other receptor classes, as outlined in the Jak/Stat Utilization Table. Researchers have found Stat3 and Stat5 to be constitutively activated by tyrosine kinases other than Jaks in several solid tumors

The Jak/Stat pathway mediates the effects of cytokines, like erythropoietin, thrombopoietin, and G-CSF, which are protein drugs for the treatment of anemia, thrombocytopenia, and neutropenia, respectively. The pathway also mediates signaling by interferons, which are used as antiviral and antiproliferative agents. Researchers have found that dysregulated cytokine signaling contributes to cancer. Aberrant IL-6 signaling contributes to the pathogenesis of autoimmune diseases, inflammation, and cancers such as prostate cancer and multiple myeloma. Jak inhibitors currently are being tested in models of multiple myeloma. Stat3 can act as an oncogene and is constitutively active in many tumors. Crosstalk between cytokine signaling and EGFR family members is seen in some cancer cells. Research has shown that in glioblastoma cells overexpressing EGFR, resistance to EGFR kinase inhibitors is induced by Jak2 binding to EGFR via the FERM domain of the former [Sci. Signal. (2013) 6, ra55].

Activating Jak mutations are major molecular events in human hematological malignancies. Researchers have found a unique somatic mutation in the Jak2 pseudokinase domain (V617F) that commonly occurs in polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. This mutation results in the pathologic activation Jak2, associated with receptors for erythropoietin, thrombopoietin, and G-CSF, which control erythroid, megakaryocytic, and granulocytic proliferation and differentiation. Researchers have also shown that somatic acquired gain-of-function mutations of Jak1 are found in adult T cell acute lymphoblastic leukemia. Somatic activating mutations in Jak1, Jak2, and Jak3 have also been identified in pediatric acute lymphoblastic leukemia (ALL). Furthermore, Jak2 mutations have been detected around pseudokinase domain R683 (R683G or DIREED) in Down syndrome childhood B-ALL and pediatric B-ALL.

Selected Reviews:

Beekman R, Touw IP (2010) G-CSF and its receptor in myeloid malignancy. Blood 115(25), 5131–6.
Neurath MF, Finotto S (2011) IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer. Cytokine Growth Factor Rev. 22(2), 83–9.
Sansone P, Bromberg J (2012) Targeting the interleukin-6/Jak/stat pathway in human malignancies. J. Clin. Oncol. 30(9), 1005–14.
Vainchenker W, Constantinescu SN (2013) JAK/STAT signaling in hematological malignancies. Oncogene 32(21), 2601–13.
Yu H, Pardoll D, Jove R (2009) STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer 9(11), 798–809.

– See more at: http://www.cellsignal.com/contents/science-pathway-research-immunology-and-inflammation/jak-stat-signaling-pathway/pathways-il6#sthash.8SVwSWXw.dpuf

The JAK-STAT Signaling Pathway: Input and Output Integration¹

Peter J. Murray

The Journal of Immunology Mar 1, 2007; 178(5): 2623-2629 http://dx.doi.org:/10.4049/jimmunol.178.5.2623

Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.

The mammalian JAK and STAT family members have been extensively, and seemingly exhaustively, analyzed in the mouse and human systems. All four JAK and seven STAT family members have been deleted in the mouse, in addition to the creation of conditional alleles for genes whose loss of function leads to embryonic or perinatal lethality (Stat3, combined deficiency of Stat5a and Stat5b, and Jak2). In humans, detailed genetic studies have been performed in people bearing mutant Jak or Stat genes. Specific Abs to phospho-forms of each protein are used to study how the JAK-STAT cascade is activated by cytokine receptors. Crystallographic studies have illuminated structural information for multiple STAT family members in different forms. Pharmacological inhibitors have been developed for clinical use where JAK-STAT signaling is implicated in disease pathology and progression. Finally, in most cases, a specific JAK-STAT combination has been paired with each cytokine receptor, and this information translated into cell-type specific patterns of cytokine responsiveness and gene expression.

Major questions remain concerning how the JAK-STAT cascade functions to control specific gene expression patterns, and how the cascades are regulated. I will describe three elements of JAK-STAT signaling that require experimental investigation. First, I will address an unexpected experimental complication that arises from the analysis of mice and cells that lack one or more STAT family member. Second, I will use JAK1-STAT3 signaling from the IL-10R and IL-6R systems to illustrate that we lack detailed understanding of how specificity in gene expression is generated by receptors that use identical JAK-STAT members. Third, we have yet to explain how STAT activation is negatively regulated. Although the suppressor of cytokine signaling (SOCS)³ proteins are the best understood negative regulators of the JAK-STAT pathway, the biochemical mechanism of SOCS-mediated inhibition is unexplained. Moreover, additional inhibitory pathways have also been proposed to block the production of activated STATs. Collectively, I will argue that our understanding of the pathway from cytokine receptor to gene expression profile is in its infancy, but remains one of the best opportunities to dissect signal transduction.

Overview of the proximal JAK-STAT activation mechanism

The current model of JAK-STAT signaling holds that cytokine receptor engagement activates the associated JAK combination, which in turn phosphorylates the receptor cytoplasmic domain to allow recruitment of a STAT, which in turn is phosphorylated, dimerizes and moves to the nucleus to bind specific sequences in the genome and activate gene expression. Cytoplasmic domains of cytokine receptors associate with JAKs via JAK binding sites located close to the membrane (1). The postulated role of JAKs in trafficking or chaperoning the receptors to the cell surface is debated (2, 3, 4, 5, 6). Regardless of the when and where cytokine receptors and JAKs associate, their close apposition at the membrane is required to stimulate the kinase activity of the JAK following cytokine binding. At this stage in the activation of the pathway, we understand next to nothing about the structural basis of the JAK-receptor interaction, how receptor intracellular domains reorient upon cytokine binding and physically contact the JAK to receive the phosphorylation modification.

JAK-mediated phosphorylation of the receptor creates binding sites for the Src homology 2 (SH2) domains of the STATs. STAT recruitment is followed by tyrosine, and in some cases, serine phosphorylation on key residues (by the JAKs and other closely associated kinases) that leads to transit into the nucleus. This brief summary of the activation of the JAK-STAT pathway omits numerous unresolved details: the STAT monomer to dimer transition has been questioned, as has the role of phosphorylation in dimerization and nuclear transit (7). Furthermore, it is unclear how many configurations of STAT homo- and heterocomplexes are present in cells before, during, and after cytokine stimulation (8, 9,10). We do not understand the detailed structural basis for the preference of one SH2 domain for a given receptor, and we have little knowledge of how other non-JAK kinases are recruited to the receptors and phosphorylate the STATs.

Many receptors signal through a small number of JAKs

Cytokine receptors signal through two types of pathways: the JAK-STAT pathway and other pathways that usually involve the activation of the MAP kinase cascade. Although the latter will not be discussed here, it is worth noting that elegant genetic studies have demonstrated the importance of these pathways in various pathological systems (11, 12,13, 14). There are now ∼36 cytokine receptor combinations that respond to ∼38 cytokines (counting the type I IFNs as one because they all signal through the IFN-αβR). Different cells and tissues express distinct receptor combinations that respond to cytokine combinations unique to the microenvironment or systemic response of the organism. Hence, at any given time, a single cell may integrate signals from multiple cytokine receptors. Genetic studies have established that the cytokine receptor system is restrictive in that different classes of receptors preferentially use one JAK or JAK combination (7): receptors required for hemopoietic cell development and proliferation use JAK2, common γ-chain receptors use JAK1 and JAK3 whereas other receptors use only JAK1 (Fig. 1⇓). Unexplained is the selective use of these combinations: why the IFN-γR rigidly uses the JAK1, JAK2 combination is unknown as is the restricted use of TYK2. Compared with JAK1–3, TYK2 is unusual in that loss of function mutations in the mouse have shown obligate, but not absolute, requirements in IFN-αβR and IL-12R signaling (15, 16). In contrast, human TYK2 seems to be essential for signaling through a broader range of cytokine receptors (17).

FIGURE 1.

The majority of cytokine receptors use three JAK combinations. Shown are well-studied cases where JAK usage by each cytokine receptor has been established by genetic and biochemical studies. Exceptions shown are the G-CSFR (∗) where it is currently unclear whether both JAK1 and JAK2 are required together. Additionally, the IL-12R (†) and IL-23R (†) require TYK2 but the requirement for JAK2 has not been definitively determined. Receptors that use JAK2 and JAK3, JAK3 alone, TYK2 alone, or JAK3 and TYK2 have not been described.

The preferential association of JAKs to certain receptor classes raises several issues. First, how did the JAK-receptor combinations evolve? Because the number of receptors is relatively large, why has the number of JAKs remained small? Why have the combinations of JAK pairs also remained small given that there are 10 possible combinations that can be used (Fig. 1⇑)? Second, how flexible is the cytokine receptor-JAK pair? That is, can receptors be engineered for interchangeable JAK use, or is a given JAK combination fixed for a specific receptor class? For example, can JAK1, JAK3, or TYK2 activate erythropoietin receptor (EpoR) signaling (if so engineered) or is JAK2 obligatory for signaling? These questions allude to a fundamental issue that concerns the function of the JAK in cytokine receptor activation: if the only function of the JAKs is to phosphorylate tyrosine resides on the cytoplasmic domain of the receptors, then it should be possible to trade JAK-receptor pairs. If these receptors retain identical downstream gene expression profiles, then the signal generated by the JAK is generic and functions primarily to activate the receptor (6). Conversely, it is also possible that each receptor-JAK combination retains crucial specificity functions and swapping, for example, JAK1 for JAK2 on the EpoR will modify or destroy a specific function in erythrogenesis. These questions can be addressed experimentally by replacing one preferred JAK binding site for another in genes encoding different receptors. The EpoR is a good test example because the activity of the receptor and its signaling pathway is essential for life and erythropoiesis is readily assayed.

Core versus cell-type specific STAT signaling

Microarray experiments designed to monitor changes in gene expression induced by JAK-STAT signaling have revealed that both cell-type specific transcription and core, or stereotypic, mRNA profiles are induced by activated cytokine receptors in different cell types (Fig. 2⇓). For example, IFN-γ, via STAT1, induces the expression of a similar cohort of genes regardless of the cell type tested (18). These genes are often termed the “IFN signature” and overlap with the gene expression pattern induced by IFN-αβ signaling that also involves STAT1, in cooperation with STAT2 and IRF9. The IFN signature is readily observed in microarray experiments and is indicative of STAT1 activity. The STAT6 pathway activated by IL-4 or IL-13 provides an example of a cell-type specific response. IL-4-regulated genes in T cells have a distinct signature compared with IL-4/IL-13 signaling in macrophages or other non-lymphocytes (19, 20, 21, 22). In the latter, genes such as Arg1(encoding arginase 1) are often induced >100-fold but are silent in T cells (23, 24, 25, 26,27). Collectively these data argue that STATs activate defined gene sets, depending on their genomic accessibility, and possibly on cofactors that further refine gene expression profiles. STAT3 signaling illustrates a more complex system and will be discussed below to illustrate the distinctions between IL-6 and IL-10 signaling.

FIGURE 2.

Core signaling by STATs. Representative examples of gene expression induced by STAT signaling in different tissues. The examples were extracted and edited from numerous microarray and empirical studies.

Interpreting experiments using STAT loss-of-function systems

Experiments with the different STAT knockout mice, and cells derived from these animals, have been critical for understanding specific requirements of individual STATs in gene expression following cytokine receptor signaling. The interpretation of these experiments is generally straightforward. For example, STAT5a and STAT5b are essential for the expression of genes that promote hemopoietic survival (28, 29, 30) whereas STAT1 is required for the expression of IFN-regulated genes that are involved in the protection against pathogens (18). However, by EMSA and immunoblotting experiments, most cytokines have been shown to activate multiple STATs, prompting experiments to determine transcriptional responses that can be activated in the absence of a given STAT. An initial example of this type of approach was performed by Schreiber and colleagues who interrogated gene expression profiles induced by IFN-γ signaling in the absence of STAT1 (31, 32). In these experiments, IFN-γ was used to stimulate STAT1-deficient bone marrow-derived macrophages and fibroblasts. Numerous genes were induced by IFN-γ in the absence of STAT1, leading to the conclusion that the IFN-γR activates a STAT1-independent gene expression program. However, inspection of the genes induced by IFN-γ signaling in STAT1-deficient cells shows many to be STAT3-regulated genes such asSocs3, Gadd45, and Cebpb. STAT3 phosphorylation is normally induced by IFN-γ in wild-type cells but in the absence of STAT1, STAT3 signaling is dominant. What is the mechanism of this effect? We now know from experiments using STAT-deficient cells that receptor occupancy, or lack of occupancy by the dominant STAT that binds the receptor, causes a switch from one activated STAT to another (33). A converse example is the conversion of IL-6R signaling to a dominant STAT1 activation in STAT3-deficient cells (34). This switch causes the downstream induction of the IFN gene expression pathway just as IFN-γ would cause in wild-type cells.

A related example is observed when IL-6 signaling is tested in the absence of SOCS3. SOCS3 is induced by STAT signaling from different cytokine receptors and functions as a feedback inhibitor of the IL-6R (and the G-CSFR, LIFR, and leptinR) by binding to phosphorylated Y757 on the gp130 cytoplasmic domain (see below). However in the absence of SOCS3, STAT3 phosphorylation is greatly increased (35, 36, 37). At the same time however, STAT1 phosphorylation is also induced, leading to a dominant IFN-like gene expression signature (35, 36). Thus SOCS3 regulates both the quantity and type of STAT signal generated from the IL-6R. Although the mechanism of the SOCS3 effect is unclear, the promiscuity of different receptors for different STATs argues that loss-of-function experiments must be carefully examined for the activation of other STAT molecules that fill the “hole” created by the loss of one STAT. These data also suggest that different cytokine receptors have evolved selectivity for different classes of STATs. Although STAT1 and STAT3 can apparently interchangeably bind the IL-6R or IFN-γR when either molecule is missing, signaling in wild-type cells shows a strong preference for one STAT over the other. Likewise, other receptors may have evolved to bind only one STAT, and in the absence of the key STAT, the other STATs cannot bind and/or be activated by the receptor.

The above examples primarily describe experiments using STAT1–STAT3-activating receptors but these are not isolated cases. In T cells stimulated by IL-12, STAT4 is activated and drives IFN-γ production. This pathway is a central regulatory event in the development of the Th1 type T cell responses. IFN-αβ, via the IFN-αβR, also activates STAT4 (in addition to STAT1 and STAT2 that forms a complex with IRF-9 to mediate anti-viral gene expression) but cannot activate strong IFN-γ production and therefore cannot drive Th1 development (38). However, in the absence of STAT1, IFN-αβ causes a large increase in IFN-γ production, especially in vivo during viral infection (39, 40). These data were originally interpreted to mean that STAT1 normally suppressed IFN-γ production. However, the data can just as easily be resolved when we consider that STAT4 activation from the IFN-αβR is increased in the absence of STAT1. Recent data confirm this interpretation but also show that STAT4 activation by the IFN-αβR, although increased, cannot sustain IFN-γ production from T cells when compared with IL-12 (38). This is probably because of the stronger differential activity of SOCS1 on the IFN-αβR versus the IL-12R (discussed below). I would predict that an IFN-αβR that is refractory to SOCS1 (or active in a Socs1^−/− background) would behave identically to the IL-12R in the absence of STAT1.

Although loss of gene expression may be observed in a given STAT knockout, a corresponding increase in the ectopic activation of another STAT pathway may confound the interpretation of results in both in vitro and in vivo systems. Because specific Abs are available for each tyrosine-phosphorylated STAT molecule, a simple solution is to first measure which other STATs are activated by a given receptor in the absence of the STAT of interest. Experiments using STAT knockout systems should also be supported by additional data that uses complimentary mutations in the receptor that ablate STAT recruitment, or complete loss of the receptor. Finally, it is worth noting that the loss of a STAT pathway from a receptor signaling system can cause additional loss of key negative regulatory systems including feedback loops such as SOCS induction as presently debated for G-CSFR signaling and receptor systems discussed below (41, 42, 43, 44, 45).

FIGURE 3.

Proposed differential STAT activation by IL-10 or IL-6. Shown are three classes of genes activated by STAT3 where Socs3 is a representative “common” gene induced by both receptors. In the absence of SOCS3, the IL-6R can activate the anti-inflammatory genes in the same way as the IL-10R. The mechanism of this effect remains to be established.

JAK/STAT Activation Inhibitors

The JAK/STAT pathway plays an important role in cytokine receptor-mediated signal transduction via activation of downstream signal transducers and activators of transcription (STAT), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) pathways.
These inhibitors are useful tools for exploring the contribution of JAK/STAT-mediated signaling.

JAK/STAT Activation Inhibitors

AG490	JAK2 inhibitor	10 mg
AZD1480 NEW!	JAK1 & JAK2 inhibitor	5 mg
CP-690550	JAK3 Inhibitor	5 mg
CYT387 NEW!	JAK1/JAK2 & TBK1/IKK-ε inhibitor	10 mg
Ruxolitinib	JAK1 & JAK2 Inhibitor	5 mg

Methotrexate Is a JAK/STAT Pathway Inhibitor

Sally Thomas, Katherine H. Fisher, John A. Snowden, Sarah J. Danson, Stephen Brown, Martin P. Zeidler

Published: July 1, 2015
DOI: http://dx.doi.org:/10.1371/journal.pone.0130078

Background

The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains.

Methods & Findings

We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin.

Conclusions

Aminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, we suggest that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential.

Citation: Thomas S, Fisher KH, Snowden JA, Danson SJ, Brown S, Zeidler MP (2015) Methotrexate Is a JAK/STAT Pathway Inhibitor. PLoS ONE 10(7): e0130078. http://dx.doi.org:/10.1371/journal.pone.0130078

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Blocking Differentiation to Produce Stem Cells

Posted in Biological Networks, Gene Regulation and Evolution, Bone marrow derived cells, Cancer - General, Cell Biology, Signaling & Cell Circuits, Clinical & Translational, Clinical Genomics, Curation, Developmental biology, Gene Regulation, Genome Biology, Hematopoiesis, Innovations, Regenerative Biology and Medicine, Stem Cells for Regenerative Medicine, tagged Adult stem cell, blocking cell differentiation on November 19, 2015| Leave a Comment »

Blocking Differentiation to Produce Stem Cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Blocking Differentiation is Enough to Turn Mature Cells into Stem Cells

November 18, 2015 by mburatov

https://beyondthedish.wordpress.com/2015/11/18/blocking-differentiation-is-enough-to-turn-mature-cells-into-stem-cells/

Hiroshi Kawamoto led a collaboration between the RIKEN Center for Integrative Medical Science and other institutions in Japan and Europe that examined the possibility that adult cells can be maintained in a stem cell-like state where they can proliferate without undergoing differentiation. They discovered that in immune cells, blocking the activity of one transcription factor can maintain the cells in a stem cell-like state where they continue to proliferate and still have the capacity to differentiate into different mature cell types.

Kawamoto and his team genetically engineered hematopoietic progenitor cells from mice to overexpress the Id3 protein. Id3, or inhibitor of DNA binding 3, is an inhibitory protein that forms nonfunctional complexes with other transcription factors. In particular, Id3 inhibits so-called “E-proteins,” (such as TCF3) which drive the progenitor cells to differentiate into immune cells.

Overexpression of Id3, in addition to soaking the cells in a cocktail of cytokines, cause the cells to continue to divide as stem cells. However, when the cytokines were withdrawn, the cells differentiated into various types of immune cells.

Next, Kawamoto and his collaborators infused these engineered hematopoietic progenitors into mice that had been depleted of white blood cells. They discovered that their Id3-overexpressing cells could expand and replenish the white blood cell population of these.

In a follow-up experiment, Kawamoto and his crew recapitulated this experiment using human umbilical cord blood hematopoietic progenitors. Just like their mouse counterparts, these umbilical cord cells could be maintained in culture, and then, upon change of culture conditions, could differentiate into blood cells.

Because these cells can be kept in an undifferentiated state and can extensively proliferate, this culture system provides a model for studying the genetic and epigenetic basis of stem cell self-renewal. And it might also allow scientists to inexpensively grow large quantities of immune cells for regenerative medicine or immune therapies.

This work was published in Stem Cell Reports, October 2015 DOI: http://dx.doi.org:/10.1016/j.stemcr.2015.09.012.

ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein [ Homo sapiens (human) ]

Gene ID: 3399, updated on 15-Nov-2015

http://www.ncbi.nlm.nih.gov/gene?Db=gene

Official Symbol ID3 provided by HGNC

Official Full Name inhibitor of DNA binding 3, dominant negative helix-loop-helix protein provided by HGNC

Primary source HGNC:HGNC:5362 See related Ensembl:ENSG00000117318; HPRD:02608; MIM:600277; Vega:OTTHUMG00000003229

Gene type protein coding

RefSeq status REVIEWED

OrganismHomo sapiens

LineageEukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo

Also known as HEIR-1; bHLHb25

Summary The protein encoded by this gene is a helix-loop-helix (HLH) protein that can form heterodimers with other HLH proteins. However, the encoded protein lacks a basic DNA-binding domain and therefore inhibits the DNA binding of any HLH protein with which it interacts. [provided by RefSeq, Aug 2011]

Orthologs mouse all

Location:: 1p36.13-p36.12

Exon count:: 3

Annotation release	Status	Assembly	Chr	Location
107	current	GRCh38.p2 (GCF_000001405.28)	1	NC_000001.11 (23557930..23559794, complement)
105	previous assembly	GRCh37.p13 (GCF_000001405.25)	1	NC_000001.10 (23884421..23886285, complement)

Chromosome 1 – NC_000001.11 Genomic Context describing neighboring genes

Related articles in PubMed

The potential role of inhibitor of differentiation-3 in human adipose tissue remodeling and metabolic health.Svendstrup M, et al. Mol Genet Metab, 2014 Nov. PMID 25239768
A functionally significant polymorphism in ID3 is associated with human coronary pathology.Manichaikul A, et al. PLoS One, 2014. PMID 24603695, Free PMC Article
ID3 mutations are recurrent events in double-hit B-cell lymphomas.Gebauer N, et al. Anticancer Res, 2013 Nov. PMID 24222112
ID3 contributes to cerebrospinal fluid seeding and poor prognosis in medulloblastoma.Phi JH, et al. BMC Cancer, 2013 Jun 15. PMID 23768125, Free PMC Article
Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing.Richter J, et al. Nat Genet, 2012 Dec. PMID 23143595

Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

Tomokatsu Ikawa, Kyoko Masuda, Mirelle J.A.J. Huijskens, Rumi Satoh, Kiyokazu Kakugawa, Yasutoshi Agata, Tomohiro Miyai, Wilfred T.V. Germeraad, Yoshimoto Katsura, Hiroshi Kawamoto

Stem Cell Reports Nov 10, 2015; Volume 5, Issue 5, 716–727. DOI: http://dx.doi.org/10.1016/j.stemcr.2015.09.012

Highlights

•Overexpression of ID3 endows hematopoietic progenitors with self-renewal activity
•A simple block of cell differentiation is sufficient to induce stem cells
•Induced leukocyte stem (iLS) cells exhibit robust multi-lineage reconstitution
•Equivalent progenitors were produced from human cord blood hematopoietic stem cells

Self-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status. We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy.

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2040173852/2053709392/fx1.jpg

Somatic tissues with high turnover rates, such as skin, intestinal epithelium, and hematopoietic cells, are maintained by the activity of self-renewing stem cells, which are present in only limited numbers in each organ (Barker et al., 2012,Copley et al., 2012, Fuchs and Chen, 2013). For example, the frequency of hematopoietic stem cells (HSCs) in the mouse is about 1 in 10⁵ of total bone marrow (BM) cells (Spangrude et al., 1988). Once HSCs begin the differentiation process, their progeny cells have hardly any self-renewal capacity, indicating that self-renewal is a special feature endowed only to stem cells.

Cells such as embryonic stem (ES) cells that retain self-renewal potential and multipotency only in vitro can also be included in the category of stem cells. Such stemness of ES cells is thought to be maintained by formation of a core transcriptional network and an epigenetic status unique to ES cells (Lund et al., 2012, Meissner, 2010, Ng and Surani, 2011). A stem cell equivalent to ES cells, called induced pluripotent stem (iPS) cells, can be produced from somatic cells by overexpression of only a few specific transcription factors (OCT3/4, SOX2, KLF4, and C-MYC), which are thought to be the essential components in forming the core network of transcriptional factors that define the status of ES cells (Takahashi et al., 2007, Takahashi and Yamanaka, 2006, Yamanaka, 2012). It is thus generally conceived that acquisition of such a network for a somatic cell depends on the reprogramming of the epigenetic status of that cell.

On the other hand, it could be envisioned that the self-renewing status of cells represents a state in which their further differentiation is inhibited. It is known, for example, that to maintain ES/iPS cells, factors such as leukemia inhibitory factor and basic fibroblast growth factor, for mouse and human cultures, respectively (Williams et al., 1988, Xu et al., 2005), are required, and these factors are thought to block further differentiation of the cells. In this context, it has previously been shown that systemic disruption of transcription factors essential for the B cell lineage, such as PAX5, E2A, and EBF1, leads to the emergence of self-renewing multipotent hematopoietic progenitors, which can be maintained under specific culture conditions (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008). It has recently been shown that the suppression of lymphoid lineage priming promotes the expansion of both mouse and human hematopoietic progenitors (Mercer et al., 2011, van Galen et al., 2014). Therefore, it would seem theoretically possible to make a stem cell by inducing inactivation of these factors at particular developmental stages. Conditional depletion of PAX5 in B cell lineage committed progenitors, as well as mature B cells, resulted in the generation of T cells from the B lineage cells (Cobaleda et al., 2007, Nutt et al., 1999, Rolink et al., 1999). These studies, however, were mainly focused on the occurrence of cell-fate conversion by de-differentiation of target cells. Therefore, the minimal requirement for the acquisition of self-renewal potential remains undetermined.

Our ultimate goal is to obtain sufficient number of stem cells by expansion to overcome the limitation of cell numbers for immune therapies. We hypothesize that stem cells can be produced by simply blocking differentiation. As mentioned earlier, self-renewing multipotent progenitors (MPPs) can be produced by culturing E2A-deficient hematopoietic progenitors in B cell-inducing conditions (Ikawa et al., 2004a). Because it remains unclear at which developmental stage the acquisition of self-renewing potential has occurred in the case of such a systemic deletion, we thought to develop a method in which E2A function could be inactivated and reactivated in an inducible manner. We decided to use the ID3 protein for this purpose, because it is known that ID proteins serve as dominant-negative inhibitors of E proteins (Norton et al., 1998, Sayegh et al., 2003). Here we show that the overexpression of ID3 into HSCs or hematopoietic progenitor cells (HPCs) in both mouse and human induces the stemness of the progenitors and that the cells acquire the self-renewal activity. The ID3-expressing cells can be maintained in vitro as MPPs with T, B, and myeloid lineage potentials.

Results

Jump to Section
Introduction
Results
  Generation of ID3-Transduced Hematopoietic Progenitors
  IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials
  IdHP Cells Are Multipotent at a Clonal Level
  Generation of IdHP Cells from Mouse BM
  Generation of Inducible IdHP Cells Using ID3-ER Retrovirus
  Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors
Discussion
Experimental Procedures
  Mice
  Antibodies
  Growth Factors
  Isolation of Hematopoietic Progenitors
  Retroviral Constructs, Viral Supernatants, and Transduction
  In Vitro Differentiation Culture System
  Cloning of mIdHP Cells
  Colony-Forming Unit in Culture Assay
  Cell Cycle Assay
  Adoptive Transfer of mIdHP and hIdHP Cells
  PCR Analysis of Igh Gene Rearrangement
  RNA Extraction and qRT-PCR
  Microarray Analysis
Author Contributions
Supplemental Information
References

Generation of ID3-Transduced Hematopoietic Progenitors

IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials

IdHP Cells Are Multipotent at a Clonal Level

Generation of IdHP Cells from Mouse BM

Generation of Inducible IdHP Cells Using ID3-ER Retrovirus

Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors

Thumbnail image of Figure 1. Opens large image

http://www.cell.com/cms/attachment/2040173852/2053709390/gr1.jpg

Identification of cellular and molecular events regulating self-renewal or differentiation of the cells is a fundamental issue in the stem cell biology or developmental biology field. In the present study, we revealed that the simple inhibition of differentiation in HSCs or HPCs by overexpressing ID proteins and culturing them in suitable conditions induced the self-renewal of hematopoietic progenitors and allowed the extensive expansion of the multipotent cells. The reduction of ID proteins in MPPs resulted in the differentiation of the cells into lymphoid and myeloid lineage cells. Thus, it is possible to manipulate the cell fate by regulating E-protein or ID-protein activities. This inducible system will be a useful tool to figure out the genetic and epigenetic program controlling the self-renewal activity of multipotent stem cells.

Previous studies have shown that hematopoietic progenitors deficient for E2A, EBF1, and PAX5 maintain multilineage differentiation potential without losing their self-renewing capacity (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008), indicating that the inhibition of the differentiation pathway at certain developmental stages leads to the expansion of multipotent stem cells. However, the MPPs were not able to differentiate into B cells because they lacked the activities of transcription factors essential for the initiation of the B lineage program. In addition, a restriction point regulating the lineage-specific patterns of gene expression during B cell specification remained to be determined because of the lack of an inducible system that regulates B cell differentiation. Here we have established the multipotent iLS cells using ID3-ER retrovirus, which can be maintained and differentiated into B cells in an inducible manner by simply adding or withdrawing 4-OHT. The data indicated the essential role of E2A for initiation of the B cell program that restricts other lineage potentials, because the depletion of 4-OHT from the culture immediately leads to the activation of E proteins, such as E2A, HEB, and E2-2, that promote B cell differentiation. This strategy is useful in understanding the cues regulating the self-renewal or differentiation of uncommitted progenitors to the B cell pathway. Analysis of genome-wide gene expression patterns and histone modifications will determine the exact mechanisms that underlie the B cell commitment process.

The iLS cells can also be generated from human CB hematopoietic progenitors. Human iLS cells exhibited differentiation potential and self-renewal activity similar to those of murine iLS cells, suggesting a similar developmental program during human B cell fate specification. Our data are consistent with a study demonstrating the critical role of the activity of ID and E proteins for controlling the status of human HSCs and progenitors (van Galen et al., 2014). This study reported that the overexpression of ID2 in human CB HSCs enhanced the myeloid and stem cell program at the expense of lymphoid commitment. Specifically, ID2 overexpression resulted in a 10-fold expansion of HSCs, suggesting that the inhibition of E-protein activities promotes the self-renewal of HSCs by antagonizing the differentiation. This raises a question about the functional differences between ID2 and ID3. Id3 seems to suppress the B cell program and promote the myeloid program more efficiently than does ID2, because the ID2-expressing HPCs appear to retain more B cell potential than ID3-expressing iLS cells (Mercer et al., 2011, van Galen et al., 2014). The self-renewal activity and differentiation potential of ID2-HPCs derived from murine HSCs in the BM seemed to be limited both in vivo and in vitro analysis (Mercer et al., 2011). In our study, the iLS cells retained more myeloid potential and self-renewal capacity during the culture. Strikingly, the multipotent iLS cells enormously proliferated (>10³-fold in 1 month) as long as the cells were cultured in undifferentiated conditions. This could be due to the functional differences among Id family genes. Alternatively, combination with additional environmental signals, such as cytokines or chemokines, may affect the functional differences of ID proteins, although any ID proteins can repress the activation of the E2A targets, such as Ebf1 and Foxo1, that are essential for B cell differentiation. ID1 and ID3, but not ID2, are demonstrated to be negative regulators of the generation of hematopoietic progenitors from human pluripotent stem cells (Hong et al., 2011). Further analysis is required to determine the physiological role of ID proteins in regulating hematopoietic cell fate. It also remains to be determined whether the ID3-ER system can be applied to human progenitors. It would be informative to compare the regulatory networks that control B cell differentiation in mouse and human.

Immune cell therapy has become a major field of interest in the last two decades. However, the required high cell numbers restrain the application and success of immune reconstitution or anti-cancer treatment. For example, DCs are already being used in cell therapy against tumors. One of the major limitations of DC vaccine therapy is the difficulty in obtaining sufficient cell numbers, because DCs do not proliferate in the currently used systems. The method of making iLS cells could be applied to such cell therapies. Taken together, the simplicity of this method and the high expansion rate and retention of multilineage potential of the cells make this cell source appealing for regenerative medicine or immune cell therapy.

In summary, we showed that an artificially induced block of differentiation in uncommitted progenitors is sufficient to produce multipotent stem cells that retain self-renewal activity. Once the differentiation block is released, the cells start differentiating into mature cells both in vivo and in vitro. Thus, this method could be applicable for establishing somatic stem cells from other organs in a similar manner, which would be quite useful for regenerative medicine. The relative ease of making stem cells leads us to conceive that a block in differentiation is essential not only in other types of artificially engineered stem cells, such as ES cells and iPS cells, but also in any type of physiological somatic stem cell. In this context, it is tempting to speculate that it could have been easy for a multicellular organism to establish somatic stem cells by this mechanism during evolution.

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Archive for the ‘Gene Regulation’ Category

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AGENDA for Oligonucleotide Therapeutics and Delivery, April 4-5, 2016, HYATT Hotel, Cambridge, MA

Oligonucleotide Therapeutics and Delivery

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Muscular dystrophy has deficient stem cell dystrophin

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ATM and ATR Are Both Required for the Presence of Human Telomerase at Telomeres

Figure 1

ATM Effect on Telomerase Recruitment Is Partially Mediated by TRF1

Figure 2

Phosphorylation of TRF1 at Serine 367 Controls TRF1 and Telomerase Localization to Telomeres

Figure 3

Stalled Replication Forks Trigger Telomerase Recruitment

Figure 4

Figure 5

Figure 6

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Biochemistry and Dysmetabolism of Aging and Serious Illness

Fig. 12

RESULTS:

CONCLUSIONS:

EXPERIMENTAL APPROACHES:

KEY RESULTS:

CONCLUSION AND IMPLICATION:

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Tool Selection

Converging Technologies

Validating Antibodies with RNAi

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The expanding class of JAK inhibitors

Innovation means understanding and responding to unmet needs

Innovative clinical trial design

Experience plus evidence equals more innovation

Pfizer’s up-to-date Xeljanz data presentation at ACR

JAK inhibition beyond RA

JAK/STAT Activation Inhibitors

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Results

Generation of ID3-Transduced Hematopoietic Progenitors

IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials

IdHP Cells Are Multipotent at a Clonal Level

Generation of IdHP Cells from Mouse BM

Generation of Inducible IdHP Cells Using ID3-ER Retrovirus

Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors

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