A census of human RNA-binding proteins

Nature Reviews Genetics | Analysis

 Analyses of post-transcriptional gene regulation and the protein factors involved have been substantially driven forward by technological advances such as next-generation sequencing and modern protein mass spectrometry. This Analysis provides a census of 1,542 manually curated RNA-binding proteins, for which the authors have investigated interactions with different classes of RNA, evolutionary conservation, abundance and tissue-specific expression.

 

Stefanie Gerstberger,^1, Markus Hafner^2, & Thomas Tuschl^1,

Abstract

 

Post-transcriptional gene regulation (PTGR) concerns processes involved in the maturation, transport, stability and translation of coding and non-coding RNAs. RNA-binding proteins (RBPs) and ribonucleoproteins coordinate RNA processing and PTGR. The introduction of large-scale quantitative methods, such as next-generation sequencing and modern protein mass spectrometry, has renewed interest in the investigation of PTGR and the protein factors involved at a systems-biology level. Here, we present a census of 1,542 manually curated RBPs that we have analysed for their interactions with different classes of RNA, their evolutionary conservation, their abundance and their tissue-specific expression. Our analysis is a critical step towards the comprehensive characterization of proteins involved in human RNA metabolism. 

Key points

• Recent advances in next-generation sequencing methods and quantitative mass spectrometry have renewed the interest in RNA biology and the genome-wide investigation of post-transcriptional gene regulatory proteins. A global census that systematically lists the number of factors involved in post-transcriptional gene regulation (PTGR) is currently not available. Here, we provide an overall summary of the proteins involved in interactions with all classes of RNAs based on our current knowledge of PTGR; this will guide future systems-wide studies of PTGR.
• RNA-binding proteins (RBPs) are evolutionarily deeply conserved, and their structural domains diversified early in evolution.
• RBPs are among the most abundant proteins in the cell and are generally ubiquitously expressed, which mirrors their central and conserved role in gene regulation.
• Only ~2% of RBPs are tissue-specific, and most of these are mRNA- and non-coding RNA-binding proteins.
• Diseases involving RBPs show characteristic phenotypes depending on the type of RNA (for example, mRNA, ribosomal RNA and tRNA) predominantly bound by the RBPs.
• Correlated expression of RBPs across developmental processes can identify factors in shared PTGR pathways.

Introduction

Post-transcriptional gene regulation (PTGR) is essential to sustain cellular metabolism, coordinating maturation, transport, stability and degradation of all classes of RNAs (Fig. 1). Mechanistically, each of these events is regulated by the formation of different ribonucleoprotein (RNP) complexes with RNA-binding proteins (RBPs) at their core. Initially, it was thought that RNA mainly served either as the template, in the form of mRNA, or as an adaptor or a structural component during protein synthesis, provided by tRNAs and ribosomal RNAs. With the discovery of catalytic RNAs and a multitude of non-coding RNA (ncRNA) species, it was recognized that RNA is a highly versatile molecule that carries out many regulatory functions in the cell, either by acting as a guide to recognize RNA sequence motifs or RNA recognition elements present in their target RNAs, or by functioning as a scaffold and assembly platform for recruiting proteins to act synergistically¹. The characterization of the proteins transiently or stably interacting with RNAs is a prerequisite for the dissection of RNA regulatory processes.

Figure 1: Overview of the main post-transcriptional gene regulation pathways in eukaryotes.

An overview is given for the biogenesis, decay and function of the most abundant RNAs: tRNAs, ribosomal RNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), mRNAs, microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and long non-coding RNAs (lncRNAs). Processes are described from left to right. Referenced gene names and complexes in the figure are listed in Supplementary information S3 (table) and within the listed references. a | tRNAs are transcribed by RNA polymerase III (Pol III); the 5′ leader and 3′ trailer sequences are removed, introns are spliced, and the ends are joined. CCA nucleotides are added to 3′ ends, and nucleotide modifications — such as methylation (M), pseudouridylation (ψ) and deamination of adenosines to inosines (I) — are introduced before tRNA aminoacylation¹⁹⁵. b | The 5S rRNA is transcribed by Pol III, whereas 28S, 18S and 5.8S rRNAs are transcribed as one transcript by Pol I. The precursor is processed by RNA exonucleases, endonucleases and the ribonucleoprotein (RNP) RNase MRP, guided by U3 small nucleolar RNP (snoRNP). Nucleotide modifications are introduced by snoRNPs. rRNAs are assembled together with ribosomal proteins into ribosomal precursor complexes in the nucleus and transported to the cytoplasm, where they mature to functional ribosomes^{92, 196, 197}. c | Most snRNAs are transcribed by Pol II, capped and processed in the nucleus. When exported to the cytoplasm, they undergo methylation and assemble with LSM proteins into small nuclear ribonucleic particles (snRNPs) in a process aided by the survival motor neuron 1 (SMN1). These snRNPs are re-imported into the Cajal body (CB) within the nucleus, where they undergo final maturation and snRNP assembly⁸¹. U6 and U6atac snRNAs are transcribed by Pol III and are alternatively processed in the nucleus and the nucleolus¹⁹⁸. Mature snRNPs form the core of the spliceosome. d | snoRNAs and small Cajal body-specific RNAs (scaRNAs) are processed from mRNA introns, capped and modified before they assemble into snoRNPs or scaRNPs in the CB. snoRNPs and scaRNPs carry out methylation and pseudouridylation of rRNAs, snoRNAs and snRNAs, or function in rRNA processing (for example, processing of U3 snoRNA)⁸¹. e | mRNAs are transcribed by Pol II, capped, spliced, edited and polyadenylated in the nucleus. Correctly matured mRNAs are exported into the cytoplasm. Regulatory RNA-binding proteins (RBPs) control correct translation, monitor stability, decay and localization, and shuttle mRNAs between actively translating ribosomes, stress granules and P bodies^{37, 141, 142, 199, 200, 201, 202}. f | miRNAs are either transcribed from separate genes by Pol II as long primary miRNA (pri-miRNA) transcripts or expressed from mRNA introns (mirtrons) and processed into hairpin pre-miRNAs in the nucleus. After transport into the cytoplasm, they are processed into 21-nucleotide-long double-stranded RNAs. One strand is incorporated into Argonaute (AGO) proteins (forming miRNA-containing RNPs (miRNPs)) and guides them to partially complementary target mRNAs to recruit deadenylases and repress translation²⁰³. g | piRNAs are ~28-nucleotides-long, germline-specific small RNAs. Primary piRNAs are directly processed and assembled from long, Pol II-transcribed precursor transcripts, whereas secondary piRNAs are generated in the 'ping pong' cycle by the cleavage of complementary transcripts by PIWI proteins. Mature piRNAs are 2′-O-methylated and incorporated into PIWI proteins. The piRNA–PIWI complexes (piRNPs) silence transposable elements (TEs) either by endonucleolytic cleavage in the cytoplasm or through transcriptional silencing at their genomic loci in the nucleus¹⁰⁷. h | Most lncRNAs are transcribed and processed in a similar way to mRNAs. Nuclear lncRNAs play an active part in gene regulation by directing proteins to specific gene loci, where they recruit chromatin modification complexes and induce transcriptional silencing or activation¹⁸⁵. Other non-coding RNAs (for example, 7SK RNA) regulate transcription elongation rates²⁰⁴ or induce the formation of paraspeckles (PS)²⁰⁵. Cytoplasmic non-coding RNAs can modulate mRNA translation²⁰⁶. i | Incorrectly processed RNAs are recognized by several complexes in the nucleus and cytoplasm that initiate and execute their degradation^{207, 208}. CPSF, cleavage and polyadenylation specificity factor; EJC, exon junction complex; hnRNP, heterogeneous nuclear RNP; NGD, no-go decay; NMD, nonsense-mediated RNA decay; NSD, non-stop decay; PABP, poly(A)-binding protein.

The recent development of large-scale quantitative methods, especially next-generation sequencing and modern protein mass spectrometry^{2, 3, 4, 5, 6}, facilitates genome-wide identification of RBPs, their protein cofactors and their RNA targets. Deep-sequencing approaches using immunoprecipitation of RBPs, with or without in vivo RNA–protein crosslinking (crosslinking and immunoprecipitation followed by sequencing (CLIP–seq) and RNA immunoprecipitation and sequencing (RIP-seq), respectively)^{2, 3}, as well as in vitro evolution methods^{7, 8}, revealed the binding ranges of RBPs and showed that many RBPs bind to thousands of transcripts in cells at defined binding sites.

 

Despite the growing amount of data collected on RBPs, many questions remain to be answered. Researchers still have an incomplete understanding of how binding specificity is achieved and how the regulatory function of an individual RBP is influenced by synergy and competition with other RBPs. We argue that a balanced approach of detailed biochemical and functional studies paired with complex systems-biology methods will ultimately lead to an understanding of the principles underlying PTGR networks.

 

Although much of the published research centres on mRNA-binding proteins (mRBPs) and messenger RNPs, PTGR is not limited to mRNA maturation and regulation; it also includes processes acting on ncRNAs. In this respect, it may not be surprising that, among the ~150 RBPs listed in the Online Mendelian Inheritance in Man (OMIM) database as being linked to human diseases, only one-third are described as directly binding mRNAs; the others target diverse ncRNAs⁹.

 

Here, we present a census of 1,542 human RBPs that interact with all known classes of RNAs, detail their families and evolutionary conservation across species, and analyse their expression across tissues and their potential roles in developmental processes. This catalogue of RBPs will guide future analyses of RBPs and provide an overview of known RNA pathways and their protein components.

 

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Conclusions

A census of human RBPs is essential for organizing our current molecular and genetic understanding of the role of RNA in general gene expression and PTGR. This catalogue provides researchers with a newly curated resource to guide their investigations of PTGR processes and to systematically study RBPs. An analogous catalogue that assesses the abundance of all expressed RNAs (that is, the RBP targets) and that classifies them across tissues and cell types is still missing. Such a catalogue would be a useful complementary document to this census.

 

Of the ~20,500 protein-coding genes in humans, we determined that 7.5% are directly involved in RNA metabolism by binding to and/or processing RNA, or by constituting essential components of RNPs. RBPs are structurally diverse and include many distinct classes of RBDs. Indeed, whereas the three most abundant DNA-binding domains account for 80% of all TFs⁵⁸, the three most abundant RBDs accounted for only 20% of all RBPs in our census. Based on target-RNA categorization, we found that nearly 50% of RBPs acted in mRNA metabolic pathways and 11% constituted ribosomal proteins, while the rest were involved in the diverse number of ncRNA metabolic processes. The target-based categorization of RBPs can assist interpretation of disease phenotypes and mutations emerging from rapidly increasing patient genome sequencing, and may guide future functional studies. When considering abundances, we found that ribosomal proteins and mRBPs were the most abundant RBPs in the cell. Nevertheless, most RBPs were ubiquitously expressed at higher levels than the residual protein-coding transcriptome, and up to 20% of the total expressed protein-coding transcripts encoded RBPs. Therefore, not only is RNA metabolism one of the most conserved cellular processes, but it also has one of the highest protein copy number demands.

 

Many details of PTGR remain to be revealed, including the dissection of newly discovered RNA regulatory processes^{1, 184, 185}. The investigation of PTGR networks is aided by the rapid development of next-generation sequencing-based methods, such as RIP- and CLIP-based methods^{2, 3, 14}, ribosome profiling¹⁸⁶, in vivo RNA secondary structure profiling^{187, 188, 189}, small and long RNA-seq^{6, 190, 191}, and 3′-end sequencing methods that profile alternative polyadenylation sites and poly(A) tail lengths^{158, 192, 193, 194}. These studies reveal an unanticipated complexity in RBP binding and targeting, and highlight the need to experimentally dissect PTGR networks in various cellular systems.

Published online 04 November 2014

 



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Macrophage subsets in atherosclerosis

Nature Reviews Cardiology

Atherosclerosis is characterized by increased accumulation of macrophages within the vessel wall. In response to stimuli such as modified lipids, cytokines, and senescent erythrocytes present in the atherosclerotic lesion, these macrophages can alter their functional phenotypes. Different macrophage subsets can influence the growth and composition of the atherosclerotic plaque in distinct ways. In this Review, Chinetti-Gbaguidi et al. highlight the diverse range of macrophage phenotypes present in atherosclerotic lesions, and their roles in both plaque progression and stability.

 

Giulia Chinetti-Gbaguidi,^1, Sophie Colin^1, & Bart Staels^1,

Abstract:

Macrophage accumulation within the vascular wall is a hallmark of atherosclerosis. In atherosclerotic lesions, macrophages respond to various environmental stimuli, such as modified lipids, cytokines, and senescent erythrocytes, which can modify their functional phenotypes. The results of studies on human atherosclerotic plaques demonstrate that the relative proportions of macrophage subsets within a plaque might be a better indicator of plaque phenotype and stability than the total number of macrophages. Understanding the function of specific macrophage subsets and their contribution to the composition and growth of atherosclerotic plaques would aid the identification of novel strategies to delay or halt the development of the disease and its associated pathophysiological consequences. However, most studies aimed at characterizing the phenotypes of human macrophages are performed in vitro and, therefore, their functional relevance to human pathology remains uncertain. In this Review, the diverse range of macrophage phenotypes in atherosclerotic lesions and their potential roles in both plaque progression and stability are discussed, with an emphasis on human pathology.

 Key points

• Only M1 proinflammatory and M2 anti-inflammatory macrophages have been described in vitro—however, a wide spectrum of intermediate phenotypes has been identified in in vivo studies

• Various stimuli (cytokines, lipids and their derivatives, senescent cells, iron) can influence macrophage phenotypes in atherosclerotic lesions

• Macrophages with different functional phenotypes are likely to perform different roles in the development of atherosclerosis

• M1 macrophages are associated with symptomatic and unstable plaques, whereas M2 macrophages are particularly abundant in stable zones of the plaque and asymptomatic lesions

• Modulation of macrophage phenotypes might be a novel strategy for the pharmacological treatment of atherosclerosis

Introduction

The development of atherosclerosis involves activation of various cell types (including endothelial cells, smooth muscle cells, lymphocytes, monocytes, and macrophages) in the intima of the arteries, which results in a local inflammatory response.¹ An increase in circulating LDL-cholesterol levels and the subsequent accumulation of oxidized LDL in the subendothelial space triggers the recruitment and retention of monocytes and lymphocytes in the arterial wall. In the intima, monocytes differentiate into macrophages, which scavenge lipoprotein particles, and eventually become foam cells.² These macrophage-derived foam cells secrete inflammatory molecules and factors that further promote lipoprotein retention, degrade the extracellular matrix, and sustain inflammation.^{3, 4}

 

Progression of atherosclerosis is characterized by apoptosis of these resident macrophages in the lipid core of the lesion. The clearance of apoptotic cells is mediated by phagocytes, mostly macrophages, which recognize and internalize dead cells in a process termed efferocytosis.⁵ In early lesions, phagocytes readily clear apoptotic cells, avoiding further progression of atherosclerosis. In chronic, advanced lesions, however, efferocytosis is no longer sufficient to engulf all dead cells, and the gradual accumulation of apoptotic debris results in formation of a necrotic core, which triggers further inflammation, necrosis, and thrombosis.⁵ Macrophages are crucial in the maintenance of efficient efferocytosis, and thereby contribute to both resolving inflammation and preventing the formation of a necrotic core within the plaque (Figure 1).⁶

 

Figure 1: Potential role of M2 macrophages in efferocytosis within atherosclerotic plaques.

M2 macrophages localized in areas of neovascularization or outside the lipid core can phagocytose apoptotic M1 macrophages, contributing to the resolution of inflammation. If efferocytosis is insufficient, dead M1 macrophages accumulate and undergo postapoptotic necrosis, leading to the formation of a necrotic core, which contributes to plaque instability and rupture.

Novel observations have challenged these previously well-established concepts. Whereas atherosclerosis was initially considered a type 1 T helper cell (T_H1)-driven inflammatory process, the concept of heterogeneity of macrophages resident within lesions has gradually emerged over the past decade.^{7, 8} Firstly, several studies revealed that both monocytes and macrophages comprise heterogeneous cell populations that adapt their functional phenotype in response to specific microenvironmental signals and molecules.^{9, 10} These different monocyte and macrophage subtypes can be identified based on their differential expression of surface markers and chemokine receptors.^{9, 10} Secondly, the general dogma that tissue-resident macrophages are incapable of proliferating was challenged by investigators who observed macrophages in mouse lungs that proliferated independently of monocyte recruitment.¹¹ Both resident and recruited macrophages can be induced to proliferate by IL-4.¹¹ Moreover, in mice, macrophages in early atherosclerotic lesions are predominantly derived from recruited monocytes, whereas macrophage proliferation is a preponderant feature of advanced plaques and is influenced by microenvironment signals.¹² Accordingly, differences in resident macrophage phenotypes can also influence their capacity to proliferate locally, which can alter the abundance of macrophages with a given phenotype.^{11, 13}

 

In this Review, we describe and discuss the reported functional phenotypes of macrophages in atherosclerotic plaques, focusing mainly on human pathology. The correlative studies suggesting a link between such macrophage phenotypes and the structure or progression of atherosclerotic lesions will also be discussed.

Figure 2: Main macrophage subtypes found in atherosclerotic lesions.

Stimuli present in atherosclerotic lesions drive the differentiation of monocytes towards different macrophage phenotypes. a | M1 macrophages release proinflammatory cytokines. b | M(Hb), Mhem, and M2 macrophages are resistant to lipid accumulation, possess iron-handling capacities, and have anti-inflammatory effects. c | Mox macrophages display an antioxidant gene expression profile. d | M4 macrophages, like M1 macrophages, are proinflammatory, but lack the capacity for phagocytosis. Abbreviations: COX-2, cyclooxygenase; CXCL4, C-X-C motif chemokine 4; HMOX-1, haem oxygenase (decycling) 1; LDL, low-density lipoprotein; LXR, liver X receptor; MMP-7, matrix metalloproteinase-7; NFE2L2, nuclear factor (erythroid-derived 2)-like 2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; TLR, toll-like receptor; TNF, tumour necrosis factor.

Figure 3: Localization of macrophage subsets in human atherosclerotic lesions.

M1 macrophages are predominantly found in the plaque shoulder and lipid core, whereas M2 macrophages are most abundant in the adventitia and areas of neovascularization, which also contain iron deposits. Fibrous caps contain similar amounts of both macrophage subsets.

Conclusions

 Macrophages are important in the development of atherosclerotic lesions because they participate in all stages of plaque formation and progression.⁷⁷ During their lifetime, macrophages are exposed to a plethora of microenvironmental signals and stimuli (including cytokines modified lipids, senescent erythorocytes, and iron) that influence their transcriptional programme and functional phenotype. The intensity of these signals changes during plaque progression, and varies between plaque regions. Therefore, macrophages adapt their phenotype both over time and in response to their physical location, contributing to and modulating plaque progression and composition. The local accumulation of macrophage subpopulations that are highly competent in clearing apoptotic cells (such as M2 macrophages) can sustain efferocytosis, thereby contributing to the resolution of inflammation and prevention of necrotic core formation within plaques.

 

Whether the distinct macrophage phenotypes represent different stages of differentiation of a single population of phenotypically and functionally plastic resident macrophages, or are consequences of the recruitment and differentiation of specific monocyte subpopulations in the lesion, is still a matter of debate. However, despite rapid advances in this field, novel studies and research methods need to be developed to determine whether monocyte subpopulations give rise to specific macrophage phenotypes. Our current knowledge suggests that M2 macrophages, owing to their localization within human plaques and their intrinsic anti-inflammatory properties, are primarily associated with plaque stability. However, one of the most important challenges in this field will be to demonstrate a direct causative link between macrophage phenotype and the structure or progression of atherosclerotic lesions. Given that only correlation studies are feasible in humans, specific mouse models need to be developed.

 

Further identification of novel phenotypic and functional markers, and the use of novel large-scale expression profiling approaches and imaging technology, is expected to lead to an improved definition and understanding of the specific functions of the different macrophage subtypes identified within the plaque. The identification of biological stimuli that can modulate macrophage phenotypes could lead to the development of novel therapeutic approaches for the treatment of atherosclerosis.

Published online 04 November 2014

 

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Epigenetics of Trained Innate Immunity

The Scientist » News & Opinion » Daily News             

Documenting the epigenetic landscape of human innate immune cells reveals pathways essential for training macrophages.

By Ruth Williams | September 25, 2014

 

WIKIMEDIA, CHRISTOPH BOCK (MAX PLANCK INSTITUTE FOR

INFORMATICS)

Genome-wide epigenetic and transcription analyses of monocytes and macrophages have uncovered two crucial pathways driving macrophage training—a recently discovered form of innate immune memory—according to two studies published in Science today (September 25). Together with a third paper documenting the transcriptional diversity of early immune cell progenitors, the studies present the latest results from the ongoing European BLUEPRINT initiative, which aims to decipher the epigenomes of blood cells during health and disease.

 

“They did a very thorough transcriptomic and epigenomic analysis of these cells and . . . they uncover not just immunologic pathways, which would be expected, but also, interestingly, some metabolic pathways that may be important to the different immunologic phenotypes of these cells,” said Ofer Levy of Boston Children’s Hospital and Harvard Medical School who was not involved in the studies.

 

Monocytes are part of the innate immune system. They circulate in the blood, but also exit to surrounding tissues, differentiate into macrophages, and patrol the body disposing of pathogens and dead cells. Under certain conditions, macrophages can become either tolerant of pathogens or trained to react against additional infections. This training of macrophages is a recently discovered process, and aside from providing a physiological answer for some previously unexplained effects of vaccination, it also challenges the established dogma of innate immunity.

 

In humans, the immune system has two arms: innate and adaptive. The traditional view is that innate immunity is broad-acting and non-specific, while adaptive immunity establishes memories for very specific pathogens, explained Christine Stabell Benn, a professor of global health at the Statens Serum Institute in Copenhagen who also did not participate in the studies. “So, if you give a vaccine against measles you induce protective immunity against measles and nothing else.” But, she added, “what we have seen in our epidemiological studies is that vaccines [also] have non-specific effects.” The Bacille Calmette-Guérin (BCG) vaccine, for example, confers protection against a variety of infections with other microorganisms—and trained macrophages appear to be responsible. The new papers, said Stabell Benn, “are now providing the molecular mechanisms behind these epidemiological observations.”

 

Mihai Netea, a professor of medicine at Radboud University in the Netherlands and an author on two of the papers, has characterized trained macrophages in the dish, in animals and in healthy people, comparing the trained phenotype to tolerant macrophages, naive macrophages (neither trained not tolerant), and monocytes. But to get the bigger picture of what defines these different yet related cells, “I went to the group of Hank [Hendrik] Stunnenberg and asked for his help with the epigenetics,” Netea said. Stunnenberg is a professor of molecular biology at Radboud University, an author on all three papers and the coordinator of the BLUEPRINT consortium.

 

Netea, Stunnenberg, and their colleagues collected monocytes from healthy people and from them derived the three macrophage types—tolerant, trained, and naive. In these four cell types, they then analyzed genome-wide distributions of four epigenetic indicators of gene activity: DNAse hypersensitivity and three different histone modifications—trimethylation of histone H3 at lysine 4, monomethylation of histone H3 at lysine 4, and acetylation of histone H3 at lysine 27. They also analyzed genome-wide transcription and transcription factor binding.

 

Together the analyses pointed to specific genes and pathways that defined the four cell types, as well as the genes’ surrounding regulatory regions. Of particular interest was the discovery that genes associated with signaling via cyclic adenosine monophasphate (cAMP)—a molecule regulating cell metabolism, among other processes—and glycolysis—a pathway that produces energy from glucose—were specifically activated in the trained macrophages.

 

The team went on to show that these two pathways were necessary for developing the trained phenotype. Cultured monocytes in which cAMP signaling or the glycolysis pathway were inhibited exhibited impaired production of training-induced cytokines. Inhibition of cAMP or glycolysis in mice increased susceptibility to secondary infections following trained innate immunity.

 

Both training and tolerance induction in macrophages have a number of clinical implications, explained Netea. For example, too much tolerance can cause immunoparalysis—a life-threatening complication of sepsis, he said. Such patients could be helped, added Stunnenberg, “if we could turn around a paralyzed cell and activate it.” But training “can probably also in some situations be detrimental to the host,” said Stabell Benn, by potentially causing excessive inflammation, for example. Having the epigenomic information about these cells, she added, is therefore important “in the first place, to understand what is going on, and in the second place, because it offers the potential of both down-regulating over-energetic cells but also revitalizing those that have been paralyzed.”

 

L. Chen et al., “Transcriptional diversity during lineage commitment of human blood progenitors,” Science, doi: 10.1126/science.1251033, 2014.

 

S. Cheng et al., “mTOR- and HIF-1a–mediated aerobic glycolysis as metabolic basis for trained immunity,” Science, doi: 10.1126/science.1250684, 2014.

 

S. Saeed et al., “Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity,” Science, doi: 10.1126/science.1251086, 2014.

Tags

vaccination, macrophage, innate immunity, immunology, genetics & genomics, epigenetics and disease/medicine

 

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Targeted Brain Cancer Vaccine

The Scientist » News & Opinion » Daily News            

Mouse study demonstrates the ability of a cancer vaccine targeted against a specific oncogenic mutation to elicit a protective anti-tumor immune response.

By Anna Azvolinsky | June 25, 2014

 

NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES

A vaccine targeting a mutation found in a subset of tumors, including slow-growing brain malignancies called gliomas, can induce an immune response and prevent tumor progression in mice, according to a study published today (June 25) in Nature. Michael Platten, a neuro-oncologist at the German Cancer Research Center in Heidelberg, Germany, and his colleagues have shown in a mouse model of glioma that this peptide vaccine induces a mutation-specific immune response and can fight pre-existing tumors.

 

“This is a proof-of-principle study,” said Darell Bigner, a cancer researcher and brain tumor expert at Duke University in North Carolina. “The tumor-specific peptides [used in this study] have potential as a tumor vaccine, and should be evaluated in human clinical trials.”

 

The vaccine contains a short peptide sequence of the point mutation in the isocitrate dehydrogenase type 1 (IDH1), which is found in more than 70 percent of gliomas. “We wanted to target a tumor-specific antigen, so a frequently found mutation was an obvious choice,” said Platten.

 

The goal of cancer vaccines is to boost the immune system’s ability to recognize tumors as foreign. But so far, tumor-specific vaccines, which have mostly been tested in advanced cancer patients, have generally not been found to improve survival. Vaccinating against a single tumor-associated mutation has presented researchers with a challenge, as the spectrum of mutations change as a tumor progresses.

 

Still, targeting a so-called driver mutation—one that occurs early in tumor development and is likely to be required to sustain tumor growth—may be a viable approach. The IDH1 mutation is thought to be such a driver: it’s one of the earliest mutations to arise in gliomas, and is found in the vast majority of cells within the same tumor. “In the case of gliomas, this tumor-specific antigen may be sufficient as there is no heterogeneity [within individual tumors], to our knowledge which also made this an attractive immunotherapy target,” said Platten.

 

The researchers vaccinated mice that had a humanized version of the major histocompatibility complex (MHC), a set of cell surface molecules that are necessary to mediate specific immunity against antigens, either pathogens or tumor molecules. Because the MHC system differs between mice and humans, this humanized mouse model is a better first preclinical attempt to evaluate the potential utility of an immunotherapy prior to a first-in-human study. The immune response to the vaccine was restricted to a specific type of T cell response, the CD4-positive T cell and was able to control the IDH1 mutation expressing tumors in the mice.

 

The mice used to test the vaccine developed sarcomas rather than gliomas. “Many questions on whether this vaccine will work the same way on gliomas and other tumors with IDH1 mutations remain,” said Bigner.

 

The researchers also showed that four out of 25 patients with gliomas had innate T cell responses to the IDH1 mutations of their tumors.

 

Based on these results, Platten and colleagues in Germany are now going ahead with a small clinical trial to test the safety and immunogenicity of the vaccine in newly diagnosed IDH1-mutated glioma patients. Patients will receive chemotherapy along with the vaccine. “The tumors we are targeting are rather slow-growing . . . as opposed to very aggressive tumors,” said Platten. “This is an advantage because we have a larger time window of opportunity to induce immunity and the patients are not yet immune-compromised from prior chemotherapies.”

 

“The ultimate goal would be to target gliomas with a combination of active vaccination and a tumor microenvironment-targeted therapy,” said Platten. The vaccine could, in theory, also be effective for other tumor types that harbor the IDH1 mutation.

 

“Cancer vaccination is making a major rebound. There are many exciting trials coming up based on preclinical data,” said Drew Pardoll, an immune-oncology expert at the Johns Hopkins University School of Medicine. “Combining cancer vaccines with the new [immune checkpoint-inhibiting] antibodies is one of several exciting approaches in cancer vaccination right now.”

 

T. Schumacher et al., “A vaccine targeting mutant IDH1 induces anti-tumour immunity,” Nature, doi:10.1038/nature13387, 2014.

Tags

tumor targeting, neuroimmunology, mutation, immunotherapy, glioma, cancer vaccine, cancer and brain cancer

 

 

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Blood-based tests for colorectal cancer screening

Worldwide, screening has been shown to reduce mortality and incidence of colorectal cancer. Despite its documented success, people still fail to participate and screening rates remain low in most countries. Given that patient-reported barriers include resistance to recommended fecal-based methods or endoscopy, blood-based tests have the potential to increase participation in colorectal cancer screening programmes.

by Dr Theo deVos

 

Different Stages of Colorectal Cancer

 

 

Background
Globally, colorectal cancer (CRC) is the third most common cancer in men and the second in women, with an estimated 1.36 million cases and causing an estimated 694,000 deaths in 2012 [1]. These rates are unnecessarily high since CRC is an excellent candidate for screening as evidenced by large randomized trials demonstrating reductions in mortality and incidence [reviewed in 2, 3]. Biologically, CRC usually develops slowly, going through a progression from non-cancerous polyp to cancer over a period of a decade or more. This biology readily lends itself to screening and early detection which has a significant positive impact on the effectiveness of intervention. For example, in the United States, 5-year survival is ~90 % if the tumour is confined locally when detected, ~70% if it has spread regionally, but only ~10% if distant metastases are present [4].

Colonoscopy is the predominantly recommended method for routine screening in some countries including the United States, as it enables detection and intervention in the same procedure. It is also the diagnostic follow-up for positive results of other screening tests. However, challenges with capacity and quality, financial concerns, and patient resistance have led to its lack of use as the primary screening modality in most settings. In some countries, flexible sigmoidoscopy is showing a resurgence, with reports demonstrating mortality and incidence benefits [2]. Table 1 displays a list of common CRC screening methods along with new methods coming on-line, today.

The first non-invasive tests for CRC were based on the detection of fecal occult blood (FOBT), and these have been further developed into immunological tests (FIT) using specific antibodies to detect hemoglobin. These tests are typically designed to allow patients to collect stool samples at home and ship the sample by mail to a central laboratory for testing. A newer alternative to fecal blood testing is the analysis of genetic/epigenetic markers in fecal material. This is the basis for the Cologuard test (Exact Sciences, WI, USA), a fecal DNA test recently approved by the US FDA [5]. Blood-based screening tests that measure tumour biomarkers in plasma or serum have been developed as a minimally-invasive alternative to fecal testing. DNA methylation tests based on SEPT9 have become available in Europe and are undergoing regulatory review in China. In addition, methylated SEPT9 testing is available as laboratory-developed tests (LDTs) in the USA, and a kitted version (Epi proColon®; Epigenomics AG, Germany) is currently undergoing US FDA premarket (PMA) review [6]. Another blood-based test, the ColonSentry risk test based on an expression panel is available as an LDT in the USA and in Japan.

Given the clear benefit of screening and the long standing availability of tests, the lack of participation is disappointing, and improving screening rates is a broadly accepted goal. As an example, the ‘80 by 2018’ campaign in the USA has set a goal of 80% adherence to screening guidelines by 2018 [7]. In order to meet this goal, barriers that prevent screening must be understood and overcome. There are numerous reports focused on understanding patient barriers to CRC screening. Although this is a complex issue involving costs, time, physician recommendation and several other factors, one consistent message from these studies is that the test methods themselves present barriers. Many patients are uncomfortable with all or part of the colonoscopy process and many are also uncomfortable with collecting and shipping fecal samples [8]. As a consequence, CRCs are diagnosed symptomatically in more instances than necessary, when the disease has spread beyond the primary site, resulting in greatly reduced survival rates. The availability of a screening test using a simple and common blood draw, which can be included as part of a regular check-up, has the potential to overcome some barriers and improve screening rates.

Blood-based screening
There are a number of approaches to the measurement of cancer biomarkers in the blood. The detection and quantification of circulating tumour cells represents an early approach, which was developed into a commercial system (e.g. CellSearch; Janssen Diagnostics, NJ, USA) though this analysis has not generally been used for cancer screening. Another alternative derives from the isolation and fractionation of circulating immune cells and the quantification of gene expression panels correlated with the disease by reverse-transcriptase PCR. This ‘sentinel concept’ is the basis for the ColonSentry test (GeneNews, Canada) in Table 1. A third alternative is the measurement of metabolic products by mass-spectrometry that are correlated with the presence of cancer. As an example, a commercial test (Cologic; Phenomenome, Canada) was developed based on the measurement of serum levels of GTA-446, an anti-inflammatory fatty acid. The most developed and perhaps simplest approach in this field is the measurement of cell-free genetic or epigenetic markers in plasma or serum that are highly correlated with the presence of cancer. As shown in Table 1, the methylated Septin9 biomarker and the Epi proColon® test were developed based on this approach.

Screening biomarkers in plasma and serum
The recognition that tumour DNA contains genetic and epigenetic changes that can serve as biomarkers dates back a number of decades. As reviewed recently, the list of biomarker reports for colorectal cancer grows ever longer [9]. Although numerous studies report on marker performance, the majority of studies include only a limited number of cases and controls, and only a small subset of markers have been rigorously tested in the clinical setting. Furthermore, a review of marker studies in ClinicalTrials.gov indicated very few ongoing CRC marker screening trials. Well validated markers include methylated SEPT9 described above, and the methylation of BCAT1 and IKZF1 sequences in plasma which have shown to be correlated with CRC [10] and are currently being tested in a clinical trial in Australia. There are many interesting genetic and epigenetic markers, but most await additional validation data that will support clinical utility.

Laboratory considerations for a plasma-based screening test
The basic concept outlined in Figure 1 illustrates key points associated with development of a genetic/epigenetic screening test. CRC screening from blood samples imposes rigorous demands that impact the reduction to practice for a test including: (a) high volume (millions of tests); (b) low target copy number (~1 copy per mL); (c) fragmented DNA; (d) large sample size (e.g. 3.5 mL); and (e) kitted reagents. These are discussed using the methylated Septin9 test as a case study.

Blood draw and processing
Given that screening is a high volume activity, an inexpensive and standard sample collection method is beneficial. In this case, a simple blood draw using a standard collection tube (e.g. K2EDTA plasma collection tube) is performed at the clinic or draw station. Plasma or serum is separated and if necessary they can be re-centrifuged to ensure cell-free status. The emphasis is on preparing cell-free material to limit background contamination due to lysis of nucleated cells in the blood. While this has led to the use of specialized collections tubes (Streck, NE, USA) in the field of prenatal diagnostics, these have not been widely tested for colorectal cancer screening. Cleared plasma can be tested immediately, or stored frozen for a period of time.

Nucleic acid extraction
In this step, cell-free nucleic acids are extracted from the plasma sample. While a number of commercial methods have been developed for this purpose, it remains the Achilles heel of the process. Given the wide range in target concentration, and particularly the exceptionally low copy number expected for early cancers (in the single copy per mL range) [6], as well as the fragmented nature of cell-free DNA, the extraction methods must be designed to handle large samples (e.g. 3–4 mL of plasma), and be able to isolate fragmented DNA. The use of magnetic particles for purification coupled with modified binding and wash buffers designed to capture the full range of DNA fragments has simplified the extraction, and with the development of liquid handling platforms that can process larger volumes, this step is becoming automatable. While the reduction from 3.5 mL plasma to 100 µL of DNA eluate would raise concerns for PCR inhibition, for DNA methylation tests, it is possible to reduce the wash steps because the DNA is extensively purified in the bisulfite treatment process.

Bisulfite treatment
The bisulfite treatment process is required if the target is DNA methylation-based. Recent improvements in bisulfite conversion technology have simplified the treatment. The change to ammonium bisulfite allows for liquid reagents – a key attribute for kit development. In combination with elevated temperatures, bisulfite incubation time is reduced to less than 1 hour, enabling single shift turn-around times for tests. Furthermore, the reaction can be purified using a magnetic particle extraction that takes advantage of the same particles used for the initial DNA extraction. This process can also be automated on a standard liquid handling platform to improve throughput and quality.

Real-time PCR
For genetic (mutation)-based tests, the test can be performed immediately following initial DNA extraction, though it is important to increase the stringency of DNA washes to limit the potential for PCR inhibition. In the final steps, either genetic or epigenetic markers are measured by real-time PCR. For screening applications, the target concentration dictates the conditions and interpretation of the PCR reaction. For example, in the methylated Septin9 test, the final recovered bisulfite converted template DNA is split into three wells and run in three PCR reactions. Although the PCR reaction is run as a real-time assay, the test is essentially a qualitative end point test, since a well is called positive if a PCR curve occurs at any cycle during the course of the reaction. In addition, the results of the three reactions are combined to produce a final interpretation for a patient sample. For the CE-marked Epi proColon 2.0 product, the sample is called positive if two of three wells are positive. For the Ep proColon product undergoing US FDA PMA review, the sample is called positive if any of three wells are positive. This allows for a greater emphasis on a specific test parameter – for sensitivity (any well-positive) or test specificity (two out of three wells positive).

Summary
The use of genetic and epigenetic biomarkers for cancer screening is a field still in its infancy that has great opportunities for growth. Because these biomarkers can be used as indicators of disease, they also have diagnostic and prognostic potential that will be incorporated into the clinical-decision making process. For CRC screening, test kits are already available in Europe and other countries, and are currently under review by both the US and Chinese FDA organizations. In the US, LDTs are currently marketed, and together, all progress represents significant opportunities to generate positive momentum. The introduction of simple, blood-based screening would provide a viable alternative to patients refusing or avoiding current well established methods. The convenience factors of sample collection and processing by health professionals also avoids the challenges of faulty sampling, handling, and mailing associated with at-home self-collected tests. Finally, given the extensive collection of promising biomarkers on the horizon, mechanisms are needed now to expedite clinical utilization and validation to drive further improvements in test performance.

References
1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013. Available from: globocan.iarc.fr, accessed on 12/09/2014.
2. Kuipers EJ, Rösch T, Bretthauer M. Colorectal cancer screening – optimizing current strategies and new directions. Nat Rev Clin Oncol. 2013; 10: 130–142.
3. Brenner H, Stock C, Hoffmeister M. Effect of screening sigmoidoscopy and screening colonoscopy on colorectal cancer incidence and mortality: systematic review and meta-analysis of randomised controlled trials and observational studies. BMJ 2014; 348: g2467.
4. American Cancer Society. Colorectal Cancer Facts & Figures 2014-2016. Atlanta: American Cancer Society, 2014.
5. Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, Ahlquist DA, Berger BM. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014; 370(14): 1287–1297.
6. Potter NT, Hurban P, White MN, Whitlock KD, Lofton-Day CE, Tetzner R, Koenig T, Quigley NB, Weiss G. Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma. Clin Chem. 2014; 60(9): 1183–1191.
7. National Colorectal Cancer Round Table. Tools & Resources – 80% by 2018. nccrt.org/about/80-percent-by-2018/
8. Gimeno García AZ. Factors influencing colorectal cancer screening participation. Gastroenterol Res Pract. 2012; 2012: 483417.
9. Toiyama Y, Okugawa Y, Goel A. DNA methylation and microRNA biomarkers for noninvasive detection of gastric and colorectal cancer. Biochem Biophys Res Commun. 2014; doi: 10.1016/j.bbrc.2014.08.001.
10. Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, Duesing KR, Buckley MJ, Dunne R, Beetson I, Rand KN, McEvoy A, Thomas ML, Baker RT, Wattchow DA, Young GP, Lockett TJ, Pedersen SK, Lapointe LC, Molloy PL. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer 2014; 14: 54.

The author
Theo deVos PhD
Epigenomics Inc.,
Seattle, WA 98107, USA
E-mail: theo.devos@epigenomics.com

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The Scientist> The Nutshell> WHO: TB’s Toll Worse Than Thought

A new report from the World Health Organization finds that tuberculosis has infected hundreds of thousands more people around the world than was estimated a year ago.

By Bob Grant | October 22, 2014

 

Mycobacterium tuberculosis, the bug that causes TB
WIKIMEDIA, NIAID

   Tuberculosis (TB) is causing more infections and deaths the world over than previous estimates indicated, according to a new survey released by the World Health Organization (WHO) today (October 22). The WHO’s “Global Tuberculosis Report 2014” stated that in 2013 there were 9 million new cases of TB reported in the more than 200 countries that account for more than 99 percent of the world’s TB cases.

The number of reported cases this year is 400,000 more than the WHO estimated in last year’s report, but the increased numbers may indicate improvements in diagnosis and data reporting as well as unchecked spread of the disease. “There has been some real progress, particularly in Asia, but the overall situation remains catastrophic,” Richard Chaisson, director of the Johns Hopkins Center for Tuberculosis Research in Baltimore, Maryland, told ScienceInsider. “Improvements in some countries are offset by disastrous situations in others, with MDR [multidrug-resistant] TB, HIV-related TB, and continued high rates of

missed diagnoses and deaths. The situation in Africa is particularly horrific, with TB killing more young people than any other cause.”

 

TB kills hundreds of thousands of people every year—an estimated 1.5 million people died from the disease in 2013, according to the WHO report—second only to HIV among infectious diseases.

Some infectious disease activists are criticizing the WHO report for being overly optimistic. “On the HIV side, we're doing stuff almost twice as fast in [reducing] deaths and multiple times as fast in incidence, even though TB is curable and HIV is not,” Mark Harrington, executive director of the New York City–based Treatment Action Group, which lobbies for stronger efforts to address both HIV and TB, told ScienceInsider.

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WHO, tuberculosis, TB, statistics, infectious disease, global health, epidemiology, drug resistance and disease/medicine

 

 

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