A fascinating topic: understanding how metabolism controls cell fate decisions

One scientific question that excites me the most is how changes in metabolism can alter the propensity of a cell to commit to a specific lineage. For instance, are there metabolism-dependent mechanisms that determine whether a progenitor cell commits to becoming a fat cell instead of a muscle cell? Or even more fundamentally, early on in development – could metabolic changes underlie cell fate commitments that underpin our subsequent development?

This is an emerging topic that has been embraced by several groups across the globe. Most recently, I came across aReview in Genetics & Development that deals with the exact same question: Metabolic switching and cell fate decisions: implications for pluripotency, reprogramming and development (Cliff, T & Dalton, S; 2017)

This prompted me to dig back in my library for other papers and reviews on the topic in case others out there might find it useful. Below is a non-comprehensive list that also includes several tangentially relevant inputs from the cancer field (also, note the list of papers referenced in the above review):

  1. Vander Heiden, M. et al. (2009, Science): Understanding the Warburg effect: cell proliferation
  2. Folmes et al. (2011, Cell Metabolism): Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming.
  3. Varum et al. (2011, PLoS One): Energy metabolism in human pluripotent stem cells and their differentiated counterparts.
  4. Zhang et al. (2012, Cell Stem Cell): Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal.
  5. Folmes et al. (2012, Cell Stem Cell): Metabolic plasticity in stem cell homeostasis and differentiation.
  6. Metallo, C. & Vander Heiden, M. (2013, Molecular Cell): Understanding metabolic regulation and its influence on cell physiology.
  7. Ito, K. & Suda, T. (2014, Nature Reviews Molecular Cell Biology): Metabolic requirements for the maintenance of self-renewing stem cells.
  8. Badur et al. (2015, Biotechnology Journal): Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance.
  9. Menendez, J. (2015): Metabolic control of cancer cell stemness: Lessons from iPS cells.
  10. Qian et al. (2015, Cell Stem Cell): The Dlk1-Gtl2 locus preserve LT-HSC function by inhibiting the PI3K-mTOR pathway to restrict mitochondrial metabolism.
  11. Wu et al. (2016, Cell): Cellular metabolism and induced pluripotency.
  12. Chandel et al. (2016, Nature Cell Biology): Metabolic regulation of stem cell function in tissue homeostasis and organismal ageing.
  13. Yu, J. & Cui, W. (2016, Development): Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination.
  14. Adams et al. (2016, Cell Reports): Anabolism-associated mitochondrial stasis driving lymphocyte differentiation over self-renewal.
  15. Gu et al. (2016, Cell Stem Cell): Glycolytic Metabolism Plays a Functional Role in Regulating Human Pluripotent Stem Cell State.
  16. Ho et al. (2017, Nature): Autophagy maintains the metabolism and function of young and old stem cells.
  17. Anso et al. (2017, Nature Cell Biology): The mitochondrial respiratory chain is essential for haematopoietic stem cell function.
  18. Shell, J & Rutter, J (2017, Nature Cell Biology) ➡ commentary on (2): Mitochondria link metabolism and epigenetics in haematopoiesis.
  19. Cha et al. (2017, Nature Cell Biology): Metabolic control of primed human pluripotent stem cell fate and function by the miR-200c-SIRT2 axis.
  20. Yu et al. (2017, Nature): FGF-dependent metabolic control of vascular development.
  21. Sone et al. (2017, Cell Metabolism): Hybrid cellular metabolism coordinated by Zic3 and Esrrb synergistically enhances induction of naive pluripotency.
  22. Vander Heiden, M. & DeBerardinis, R. (2017, Cell): Understanding the intersections between metabolism and cancer biology.
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“Organopoly”: combining fun and knowledge about the wonderful human body

Board_image_for_Eventbrite

What makes up a human body? What are the names of the different organs and what are their functions? What is good and bad for the body? Find out answers to all of these questions whilst playing “Organopoly” with us. Organopoly is an interactive and eye-catching educational game that introduces children and adults alike to key human biology knowledge as well as information related to lifestyle choices and disease prevention. To promote this resource to Cambridgeshire families with children 9-16-years-old, we will be running a whole-day game event at Ross Community Centre from 10 am – 4 pm on Saturday 14 October. Two games will be played in parallel with maximum 8 players per board. Each game session will last 2h30min, with the option to attend the morning or the afternoon session. Free versions of the game and additional educational resources to take home will be provided at the end.

Ross Street Community Centre is easily accessible by car or bike. A small car park and cycle racks are available in addition to free on-street parking locally. All rooms are wheelchair-accessible.

The event is free of charge, but bookings are mandatory for individual players (bookings are not required for attending family members). This event is supported by the Royal Society of Biology Regional Grant Scheme.

Watch out for us on Twitter via @RalitsaMadsen and/or #Organopoly.

A mix of papers from the past weeks: Affimer proteins might be the new antibodies, crosstalk between ERK and PI3K/AKT/mTORC1 signalling, the power of GWAS and much more

Horribly busy these days so no chance to write extensively about these, but in case they are of relevance to others, here is a list of some potentially interesting reads:

eLife: Affimer proteins are versatile and renewable affinity reagents (Tiede et al.) ➡ a potential future alternative to antibodies.  https://elifesciences.org/articles/24903

Nature: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer (Zabala-Letona et al.). Quite surprised that the mechanisms only rely on inhibitor treatment (encouraging in a way if this is “all” that is required…). Mechanism suggested to be important for proliferating cells in general. https://www.nature.com/nature/journal/vaop/ncurrent/full/nature22964.html#ref6
Scientific Reports: Inhibition of ERK1/2 restores GSK3b activity and protein synthesis levels in a model of tuberous sclerosis (Pal et al.): https://pdf.nature.com/redirect-nature?ddsId=art:10.1038/s41598-017-04528-5&originUrl=https://www.nature.com/articles/s41598-017-04528-5&contentType=pdf ➡ relevant for insulin signalling via ERK. Had a quick skim of this paper. Not entirely convinced this is robust, HEK293s + MEFs used, high concentrations of insulin, drugs etc.
Scientific Reports: Direct binding of MEK1 and MEK2 to AKT induces Foxo1 phosphorylation, cellular migration and metastasis (Procaccia et al.). https://www.nature.com/articles/srep43078.pdf
British Journal of Cancer: Akt as a target for cancer therapy: more is not always better (lessons from studies in mice) (Wang et al.). Highlights how deletion of AKT2 in the liver can lead to increased cancer incidence, thus emphasising the importance of considering tissue-specific effects when inhibiting PI3K/AKT signalling in the clinic.   https://www.nature.com/bjc/journal/vaop/ncurrent/pdf/bjc2017153a.pdf
American Journal of Human Genetics: 10 Years of GWAS Discovery: Biology, Function, and Translation (Visscher et al.) – it is a good read.
JCI Insight: Independent tissue contributors to obesity-associated insulin resistance (Kusters et al.https://insight.jci.org/articles/view/89695
Cell Metabolism: How does fat transition from white to beige? (Marc L. Reitman) http://www.cell.com/cell-metabolism/fulltext/S1550-4131(17)30353-4?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1550413117303534%3Fshowall%3Dtrue ➡ a commentary on some recent surprising findings.
Cell Metabolism: Leptin’s Physiologic Role: Does the Emperor of Energy Balance Have No Clothes? (what a title; by Jeffrey Flier and Eleftheria Maratos-Flier) http://www.cell.com/cell-metabolism/fulltext/S1550-4131(17)30302-9?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1550413117303029%3Fshowall%3Dtrue
Cell: Type 2 Diabetes Variants Disrupt Function of SLC16A11 through Two Distinct Mechanisms (Rusu et al.http://www.cell.com/cell/fulltext/S0092-8674(17)30694-3?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867417306943%3Fshowall%3Dtrue

Neoplastic changes in stem cells, nutrient acquisition strategies of mammalian cells and much, much more

After a couple of weeks in cell culture and RNAseq land, finally some time for a bit of science reading! However, not much time for writing it all up as I usually do, so here is detailed coverage of two of my recent reads. Enjoy!

Due to some unexpected results, I have had to search the stem cell literature for information on adaptive changes due to selection pressure. Came across one important paper from 2009 in Nature Biotechnology by Werbowetski-Ogilvie et al.: Characterization of human embryonic stem cells with features of neoplastic progression ➡ based on some of these points, it is really important to characterise stem cell models thoroughly, especially if you study a cancer pathway!!

  • Variant pluripotent stem cells with neoplastic changes may arise in culture and be mistaken for “better” stem cells because of higher expression of stem cell markers.
  • Paper demonstrates that such variant stem cells (2 clones studied) have acquired expression of FGFR1 and IGF1R and have become refractory to differentiation in vitroIn vivo, such cells exhibited a better capacity for teratoma formation, but were not deemed to be malignant because they didn’t metastasise (but mice kept for 6 weeks, so perhaps this could happen eventually?).

 

Nature just published a very interesting review “Nutrient acquisition strategies of mammalian cells” by Wilhelm Palm and Craigh Thompson. A few key points that I have got out of it:

  • Metabolic flexibility in cells achieved by using different nutrient sources: prepared for periods of starvation.
  • Proliferating cells: growth factor-induced increases in nutrient uptake required to support biomass accumulation. Proliferating cells need to reorganise metabolism so that nutrients are not oxidised fully to CO2 and ATP, but used in anabolic processes.
  • Bulk of ATP production in mammalian cells + generation of non-essential metabolites: glucose, fatty acids, glutamine
  • Cancer cells have changes that enable them to survive and grow in poorly vascularised environments by using extracellular nutrients, including low-molecular-mass nutrients and macromolecules as well as cellular debris.
  • Class IA PI3K and RAS signalling pathways are central regulators of cellular nutrient acquisition. Activated by various growth factor signals. Such cell-extrinsic regulation of nutrient uptake constitutes a fundamental barrier for cellular transformation. Oncogenic changes that result in hyperactivation of Ras/PI3K signalling ➡ cellular autonomy for both cell cycle entry and nutrient acquisition.
  • mTORC1 as a central coordinator of amino acid availability and cell growth. Interplay with AMPK is central to cellular metabolic homeostasis in a variable nutrient environment. Because mTORC1 is also modulated downstream of class IA PI3K, the cell is able to integrate metabolic status and growth factor signals. Activation of AKT/mTORC1 signalling defines a cellular state of high rates of glucose and amino acid uptake through transporters with concomitant suppression of lysosomal catabolism of macromolecules.
  • PI3K and Ras pathways increase glucose uptake and glycolysis. Myc increases expression of amino acid transporters. Together, Myc, Ras and PI3K stimulate ribosomal biogenesis and mTORC1-dependent translational initiation.
  • Expression of various cell surface receptors that mediate the uptake of certain nutrients (e.g. iron or cholesterol) is ultimately modulated by the very same pathways listed above.
  • Macropinocytosis: non-selective endocytic pathway for bulk ingestion of extracellular solutes. Driven by actin-based protrusions. Actin-driven protrusions are immediate downstream responses of growth factor stimulation. Accordingly cells with oncogenic mutation  in Ras pathways exhibit high levels of macropinocytosis. It remains to be established if the same is true for cells with oncogenic PI3K mutations.
  • Epithelial cells can internalise whole neighboring cells via a non-phagocytic process ➡ process known as entosis ➡ digestion of the internalised cell. This behaviour is enhanced by oncogenic mutations in Ras.
  • Autophagy is also important for tumour survival.
  • Glutamine is the second most-consumed nutrient in proliferating cells ➡ major carbon source for anaplerotic reactions that replenish TCA cycle intermediates. Glutamine also provides nitrogen for synthesis of amino acids, nucleotides and hexosamines. In vivo, tumours also replenish the TCA cycle from glucose-derived pyruvate. Some cancers also express high levels of branched-chain amino acid transferases ➡ enables them to use leucine, isoleucine and valine as nitrogen sources. These are example metabolic adaptations that give cancer cells a survival advantage.
  • Evidence for paracrine signalling based on observations that non-transformed cells often undergo phenotypic changes in the tumour microenvironment. Mechanisms exist that allow tumour cells to acquire their nutrients from neighbouring cells. Tumours can also signal to distant organs.
  • More research required to identify how signalling pathways regulate cellular nutrient uptake and what factors determine differences in nutrient usage.

 

Other bits and pieces to check out:

In Science this week: mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate (Marat et al. http://science.sciencemag.org/content/356/6341/968)

Identification and characterization of a supraclavicular brown adipose tissue in mice (JCI Insight): https://insight.jci.org/articles/view/93166 

Feng Zhang and his crew with another CRISPR tool paper in Nature: Engineered Cpf1 variants with altered PAM specificities; https://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3900.html

Hepatic Diacylglycerol-Associated Protein Kinase Cε Translocation Links Hepatic Steatosis to Hepatic Insulin Resistance in Humans (Horst et al., Cell Reports): http://www.sciencedirect.com/science/article/pii/S2211124717306770 

Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin (Waldhart et al., Cell Reports): http://www.sciencedirect.com/science/article/pii/S2211124717306836 

MYO6 Regulates Spatial Organization of Signaling Endosomes Driving AKT Activation and Actin Dynamics (Masters et al. Cell Reports): http://www.sciencedirect.com/science/article/pii/S2211124717307052

Interesting paracrine signalling mechanism: Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells (Rodvold et al. Science Signaling) http://stke.sciencemag.org/content/10/482/eaah7177 

A Nature Reviews Endocrinology review on why people should study mice at thermoneutrality: https://www.sparrho.com/item/warming-the-mouse-to-model-human-diseases/1385ea1/

A Diabetes review on the global T2D Epidemic and the importance of early β-cell failure: http://diabetes.diabetesjournals.org/content/66/6/1432

An Expanded Genome-Wide Association Study of Type 2 Diabetes in Europeans (Scott et al. Diabetes) http://diabetes.diabetesjournals.org/content/early/2017/05/25/db16-1253

 

 

Some interesting reading: bits and pieces of recent research

Nature: Blocking FSH induces thermogenic adipose tissue and reduces body fat (Liu et al.) ➡ apparently, this could be used to treat osteoporosis and obesity associated with menopause…  Looks like a nice study, but haven’t scrutinised it properly.

Nature: Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling (Brockmann et al.). Skimmed this through, and basically the group applies high-throughput technology and protein abundance as a readout for phenotypes arising from random mutagenesis in haploid cells to identify novel regulators of signal trasnduction. They identify a new mechanisms whereby Gβγ is targeted for degradation by KCTD5 to limit the ability of GPCRs to trigger PI3K/AKT activation.

Nature resource-type paper that is important for mining -omics datasets / hypothesis generation: Architecture of the human interactome defines protein communities and disease networks (Huttlin et al.) ➡ the resource is called BioPlex 2.0; this is the abstract of the paper describing what it’s all about:

“The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein– protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease1–3. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification–mass spectrometry methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering5 of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.”

Nature News & Views on the ATP-independent mitochondrial regulation of cell fate decisions in haematopoietic stem cells which was demonstrated by two independent studies recently (linked to within the news and views). It is very interesting because it is linked back to mTOR signalling and also demonstrates the importance of mitochondria in cells that are otherwise very glycolytic and don’t rely on mitochondria to generate energy. Title to search for if interested: Mitochondria link metabolism and epigenetics in haematopoiesis (Schell, J. & Rutter, J.).

Nature News & Views (Cell forces meet cell metabolism) on another interesting mechanism relating to how cell obtain their energy during the costly process of cell-cell adhesion, with link to the original paper that reported this (. Very interesting because it is relevant for understanding how cell-cell interactions link to intracellular metabolism regulation. In the described case, force exerted through E-cadherins links to AMPK activation and increased glycolysis in epithelial cells. Also remindes me of the recent paper linking PI3K activation and remodelling of the actin cytoskeleton to release Aldolase and trigger glycolysis in preparation for cell migration (by Lewis Cantley’s group). Must admit that I haven’t read the original paper discussed in this News and Views and after skimming through the quality of the figures and methods, I already noted wrong statistics (t tests instead of 1-Way ANOVA or 2-Way ANOVA!!).

eLife: Synergistic interactions with PI3K inhibition that induce apoptosis (Zwang et al.). This group looked for genes that promote breast cancer cell survival in the face of PI3K inhibition (the initial cells they use have PIK3CA H1047R and ERBB2 amplification, they then confirm in additional cell lines). They performed a shRNA-based apoptosis screen and identified PIM2ZAK, TACC1, ZFR and ZNF565 as genes whose inhibition in the presence of the p110α/δ inhibitor GDC0941 (625 nM). It is interesting to note that from the initial shRNA screen where 54 candidates were identified, only 5 were confirmed upon orthogonal validation. Goes to show the importance of validation experiments, which they also do for the 5 final candidates by overexpressing them to check that the phenotype is rescued – a method which validated 3/5 candidates. All 5 eventually validated in vivo. I like the fact that they drug concentrations are not ridiculously high (which is the case for many papers, confounding their findings), and they also test additional inhibitors in the context of the proposed interactions and find that the effect is p110α-dependent and occurs throught he canonical signalling pathway (AKT etc.).They also use z-score filtering following the initial screen to account both for the magnitude of the effect and the consistency of replicates. Another interesting thing in this paper: they use a so-called BH3 profiling assay to determine mitochondrial priming (i.e. the proportion of anti-apoptotic and apoptotic proteins). In brief, you permeabilise the cells and incubate them with increasing concentrations of synthetic BIM peptide; you then fix the cells and stain for endogenous cytochrome c, then perform flow cytometry to quantify loss of cytochrome c (measure of mitochondrial depolarisation).

FEBS Journal: The energy sensing LKB1-AMPKα1 pathway regulates IGF1 secretion and consequent activation of the IGF1R-PKB pathway in primary hepatocytes (Chen et al.) – it is a small study limited to primary hepatocytes, and the effects are not striking, but might be interesting to read anyway. Looks like metformin reduces IGF1 secretion.

EMBO News & Views covering the 2 most recent Mitofusin papers: Let’s burn whatever you have: mitofusin 2 metabolically re-wires brown adipose tissue (Scheideler, M. & Herzig, S.)

On the biophysical/hardcore biochemistry side of things, some studies looking at molecular Cas9 mechanisms:

PNAS: Mechanisms of dupex DNA destabilisation by RNA-guided Cas9 nuclease during target interrogation (Mekler et al.)

PNAS: High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding (Boyle et al.)

Also on the techy side from Jennifer Lippincott-Schwartz’ lab in Nature: Applying systems-level spectral imaging and analysis to reveal the organelle interactome (Valm et al.)

Out of general interest and haven’t read beyond abstract, but in Science this week: Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation (Zhang, X. & van den Pol, A)

Science news

Cancer Cell: A Pan-Cancer Proteogenomic Atlas of PI3K/AKT/mTOR Pathway Alterations (Zhang et al.) – an important contribution to the cancer literature; this study takes advantage of the recent completion of the data generation stage of the The Cancer Genoem Atlas (TCGA) and performs a systematic analysis of the PI3K/AKT/mTOR pathway in over 10,000 cancer, covering 32 different major cancer types. It examines mutated genes via whole-exome/genome sequencing, transcriptomics data (RNAseq) and candidate signalling data with reverse-phase protein arrays. It is a data-dense study, but I will just highlight a few points that were interesting to me. Although activities in the PI3K/AKT and mTOR pathways were highly correlated in multiple cancer, there were also instances with evidence for some decoupling. Moreover, this study provides some high-throughput functional characterisation of a larger set of activating PIK3CA mutations in two different immortalised cell lines; one caveat with this functional analysis, however, is the fact that it relies on overexpression of the mutated proteins. Nevertheless, as the setting is the same for all tested variants, it allows for direct comparisons among them to be made – and such comparisons are quite useful as there is very limited data on the functional significance of different PIK3CA mutations beyond the well-known hotspot variants. Overall, the large sample size used to generate this data provides substantial power to detect meaningful patterns that can be used to stratify variants in the clinical setting.

Nature: TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling (Wang et al.) – a key paper and really worth a read (or at least of key points as it is quite data dense!!). Probably one of the most comprehensive papers on mTORC2 that I have seen that also links it to relevant (patho)physiology. Very interesting that growth factors (incl. insulin) tip the balance between mTORC2 and mTORC1 formation, favouring mTORC2 which then drives increased AKT activation. Evidence provided that this is relevant in conditions of PI3K/AKT hyperactivation. Very interesting!! Also, noted that they use HEK293s in multiple experiments and manage to look at PI3K/AKT signalling after serum starving these for 16h.. Usually these cells exhibit hyperactivation of this pathway, but the control conditions here show no sign of this?

Science: A subcellular map of the human proteome (Thul et al.) – an important atlas-like resource, essentially an image-based map of the subcellular proteome based on transcriptomics, immunofluorescence and mass spectrometry. It is quite impressive, the data covers 12,0003 proteins using a panel of 22 human cell lines and 13,993 antibodies. Also, they have managed to get the images annotated through a Citizen Science approach via massive multiplayer game with participation from over 180,000 worldwide players! This dataset is actually quite important as it allows for more refined interaction networks to be constructed. The interactive resources can be accessed here: http://www.proteinatlas.org

Science: ATP as a biological hydrotrope (Patel et al.) – haven’t read beyond abstract, but interesting because it seems to suggest a role for ATP in protein solubilisation within cells. (Hydrotorope = amphiphilic molecules with low cooperativity and millimolar working concentrations, differentiating them from surfactants ➡ act to solubilise hydrophobic molecules in acqueous solutions).

Science Signalling: p53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM (Stewart-Ornstein, J. and Lahav, G.) ➡ this study highlights an issue that is worth keeping in mind whenever a cell biology paper is examined: signalling dynamics do differ across cell lines, hence using a single cell lines as model system for a major phenomenon might not yield results that are broadly applicable. This paper has also got some mathematical modelling for those interested in that. I must admit that I have not read it in much detail and I am unable to comment on the computational approach.

Diabetes: Mechanisms of Insulin Resistance in Primary and Secondary Non-Alcoholic Fatty Liver (Jelenik et al.) ➡ can’t usually access, but here is the abstract:

“Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance and may result primarily from increased hepatic de novo lipogenesis (PRIM) or secondarily from adipose-tissue lipolysis (SEC). We studied mice with hepatocyte- or adipocyte-specific sterol regulatory-element binding protein-1c (SREBP-1c) overexpression as models of PRIM and SEC. PRIM mice featured increased lipogenic gene expression in liver and adipose tissue. Their selective, liver-specific insulin resistance was associated with increased C18:1-diacylglycerol (DAG) content and protein kinase C (PKC)ε translocation. SEC mice had decreased hepatic ChREBP-mediated lipogenesis and featured portal/lobular inflammation along with total, whole-body insulin resistance. Hepatic mitochondrial respiration transiently increased and declined with aging along with higher muscle reactive oxygen species production. In conclusion, hepatic insulin resistance originates from lipotoxicity but not from lower mitochondrial capacity, which can even transiently adapt to increased peripheral lipolysis. Peripheral insulin resistance is prevented during increased hepatic lipogenesis, only if adipose tissue lipid storage capacity is preserved.”

Nature Cell Biology Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling (Jin et al.)

Cell Reports: Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells (Liu et al.) – interesting paper that demonstrates that one of the mechanisms whereby the core stemness factor OCT4 maintains pluripotency is by bookmarking stemness genes during mitosis, i.e. a memory mechanisms that allows for re-expression of these genes when mitosis is completed. This is important in stem cells due to their unusual cell cycle characteristics (10-12h cycling, no G0 phase and very short G1 phase). I find one of their approaches quite cool – testing the effect of specifically degrading OCT4 during mitosis by fusing OCT4 to a Cyclin destruction box.

Nature Protocols: FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry (Arrigucci et al.) note that this is only applicable for non-adherent cell types at the moment)

eLife: Addressing the ethical issues raised by synthetic human entities with embryo-like features (Aach et al.) – interesting read, I think…

 

 

 

This week’s interesting science

Nature: Common genetic variation drives molecular heterogeneity in human iPSCs (Kilpinen et al.). The first results form the large-scale HipSci initiative funded by Wellcome.

A very interesting Nature study on vascular development and how it is regulated by FGF-dependent control of metabolism (glycolysis) via Myc: FGF-dependent metabolic control of vascular development (Yu et al. 2017). Here is a copy of the final conclusion of the paper: The FGF/MYC/HK2-dependent regulation of vascular development is unexpected. Previously, FGF activity has been linked to prevention of endothelium-to-mesenchymal transition both in the lymphatic is unexpected. Previously, FGF activity has been linked to prevention of endothelium-to-mesenchymal transition both in the lymphatic16 and in the systemic vasculature17, injury response18 and maintenance of endothelium-to-mesenchymal transition both in the lymphatic and in the systemic vasculature, injury response18 and maintenance of vascular integrity19. While the FGFR1 and FGFR3 are the receptors and in the systemic vasculature17, injury response18 and maintenance of vascular integrity19. While the FGFR1 and FGFR3 are the receptors involved, which of the 22 FGF family members is responsible for the required FGF signalling input is not known. In summary, FGF signalling regulates blood and lymphatic vascular development through control of endothelial metabolism driven by MYC-dependent regula-tion of HK2 expression. Therapeutic targeting of this FGF–MYC–HK2 pathway may open new possibilities for treatment of diseases associated with insufficient or excessive vascular growth.

Just out in Nature Medicine: Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients (Zehir et al. 2017) ➡ Interesting recurrence of certain well-known oncogenes (e.g. PIK3CA) across a range of tumour types, irrespective of lineage, but also very interesting to see how mutations in certain oncogenes are absent from specific tumour histotypes. TP53 mutations top the list and correlate positively with more aggressive cancers. Most of the TP53 mutations were inactivating due to truncation or altered splicing. Second on the list is KRAS G12 followed by PIK3CA H1047R and E545K.

Cell Metabolism: PPARδ Promotes Running Endurance by Preserving Glucose (Fan et al. 2017). Just skimmed through, looks quite comprehensive. PPARd orchestrates the transcriptional switch to fat utilisation and glucose sparing in response to exercise.

Cell Metabolism: DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging (Park et al. 2017) – a mechanism that involves phosphorylation of Hsp90 and inhibition of AMPK.

An interesting paper in Cell (Stem Cell Lineage Infidelity Drives Wound Repair and Cancer by Ge et al.) that uncovers how tumour cells hijack normal wound repair processes which under normal circumstances allow for transient stem cell lineage infidelity. In contrast, a pre-cancerous stem cells are locked into this plastic state, giving rise to excessive growth and ultimately full-blown cancer.

Nature Protocols: Assessment of engineered cells using CellNet and RNA-seq (Radley et al. 2007) – a how-to-guide for the computational platform CellNet, which allows one to upload one RNAseq data from a particular cell type / stage and compare it to large datasets on different cell types. This should be very useful for estimation of cell fate transitions in response to different differentiation perturbations.

 

eLife: Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering (Lee et al. 2017). These guys test different modifications of sgRNAs and ssODN repair templates and show that they can be tolerated. For instance, they tag the ssODN with an Alexa-647 which allows them to enrich for cells that have been successfully transfected with the repair template. They also manage to fuse the sgRNA and ssODN template to each other, complex these with Cas9 (RNP), and show that this improved cellular delivery based on cationic polymers. However, note that despite this improval the efficiency of gene editing is still lower compared to nucleofection/electroporation under comparable conditions.

Nature Methods: CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets (Tsai et al. 2017). A new genome-wide method is described for the assessment of CRISPR off-target effects in vitro and following tag integration into host cell DNA. Much more sensitive compared to previous methods that achieved whole-genome assessment of off-target effects. A second study in Nature Methods reports on a different method (SITE-seq) that also aims to profile genome-wide off-target effects with high sensitivity: Mapping the genomic landscape of CRISPR–Cas9 cleavage (Cameron et al. 2017). However, the specificity of Cas9-mediated gene editing in a cell will also be highly cell-specific and depend  on the concentration of enzyme and additional components that are delivered for gene editing (sgRNA, ssODN etc.). The effect of increasing sgRNA and Cas9 concentrations in vitro is assessed in the biochemical assay reported by Cameron et al. Both of the above methods achieve a much higher sensitivity of off-target site detection because they specifically enriched for DNA fragments associated with Cas9 cleavage prior to sequencing (i.e. all the random background is removed). Once such off-targets are identified genome-wide, they can be interrogated in any cell line that has been edited – many will, however, remain intact in the cell because of the effects of chromatin on target accessibility as well as cell-specific DNA repair mechanisms.

 

The science round-up of the week

Nature & Nature++

The implications of this Nature paper from last week are major for the stem cell community: Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations (Merkle et al. 2017).

Nature Genetics: Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci (Stender et al. 2017)

Nature Genetics: Pathogenic variants that alter protein code often disrupt splicing (Soemedi et al. 2017). Copied from abstract: “We analyzed 4,964 published disease-causing exonic mutations using a massively parallel splicing assay (MaPSy), which showed an 81% concordance rate with splicing in patient tissue. Approximately 10% of exonic mutations altered splicing, mostly by disrupting multiple stages of spliceosome assembly. We present a large-scale characterization of exonic splicing mutations using a new technology that facilitates variant classification and keeps pace with variant discovery.” I can’t even imagine how expensive this study must have been…

Nature Reviews Endocrinology: Metric for glycaemic control – from HbA2c to continuous glucose monitoring. Copied from abstract so you can decide if relevant or not: “I focus on markers of average glycaemia and the utility and/or shortcomings of HbA1c as a ‘gold-standard’ metric of glycaemic control; the notion that glucose variability is characterized by two principal dimensions, amplitude and time; measures of glucose variability that are based on either self-monitoring of blood glucose data or continuous glucose monitoring (CGM); and the control of average glycaemia and glucose variability through the use of pharmacological agents or closed-loop control systems commonly referred to as the ‘artificial pancreas’. I conclude that HbA1c and the various available metrics of glucose variability reflect the management of diabetes mellitus on different timescales, ranging from months (for HbA1c) to minutes (for CGM).”

Nature Cell Biology: Cell Competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes (Kon et al. 2017). This very interesting for the benign-malignant paradox that refers to the occurrence of cancerous mutations in benign disease without further progression to cancer. Here is a copy of the abstract (I read the paper, and it seems solid experimentally although they perform all the wrong statistics throughout!!): “Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.” There is a news & and views as well: “Metabolic changes promote rejection of oncogenic cells.”

Nature Cell Biology: Metabolic control of primed human pluripotent stem cell fate and function by the miR-200c-SIRT2 axis (Cha et al. 2017). After a quick skim, seems OK overall, except that I don’t trust the qPCR results because of the way they are represented. Would be great if people could stop normalising to samples where a gene is not expressed anyway (we have all been taught basic maths and know that you can’t divide by 0)… Not an attack on this study only, but on most qPCR studies these days. There is also a news&views on this paper in Nature Cell Biology as well (SIRT2 and glycolytic enzyme acetylation in pluripotent stem cells). What is interesting, though, is this whole concept of a glycolytic-acetyl-coA switch that could interact with the epigenetics of stem cells and their cell fate decisions.

Nature Cell Biology: SWELL1 is a regulator of adipocyte size, insulin signalling and glucose homeostasis. Can’t actually access the actual article 😦 It says aop, not sure what it means, though.

Cell Metabolism 

For those of us with a sweet tooth (a pathological tendency to snack!!),  FGF21 Is a Sugar-Induced Hormone Associated with Sweet Intake and Preference in Humans (Søberg, Sandholt et al. 2017). The gist of it is that there are human genetic variants in FGF21 that increase sweet preference, but surprisingly they do not correlate with obesity/diabetes. I found some of the points in the Introduction interesting – those regarding the difference between mouse and human FGF21 biology. So a word of caution when it comes to metabolism and species-specific differences!

From the Japanese iPSCs experts on the effect of metabolism on pluripotency states, published in Cell Metabolism: Hybrid Cellular Metabolism Coordinated by Zic3 and Esrrb Synergistically Enhances Induction of Naive Pluripotency (Sone et al. 2017). Currently, a hot topic in the stem cell field is the notion of naive vs primed pluripotency. Human iPSCs and ESCs are thought to be “primed”, i.e. more lineage-restricted and corresponding to the post-implantation epiblast state, compared to mouse counterparts which correspond to pre-implantation embryonic cells. Mouse ESCs can be induced to become even more naive (so can human ESCs/iPSCs as of recently) with certain small molecules or by genetic perturbation. Interestingly, whereas more primed stem cells rely on glycolysis, and low rates of oxidative phosphorylation, naive pluripotency seems to require both glycolysis and oxidative phosphorylation. This balance is now shown to depend on the synergy between the transcription factors Zic3 and Esrrb which both activate glycolysis genes, but have opposite effects on OXPHOS. It is really interesting to follow the metabolic theme in stem cells – the ability of cells to shift their metabolism is emerging as an important prerequisite for appropriate cell fate regulation. Also, although this study determines that both glycolysis and OXPHOS need to be ON for efficient reprogramming of mouse fibroblasts to naive stem cells, it still doesn’t have the answer as to why this is the case. Technically, this study is also quite neat – done well! Although quite interesting to see raw Ct values being reported as opposed to log-transformed derivatives (BUT great that the spread of the data is shown properly and SDs are used!).

A review on “Autophagy and Tumour Metabolism” (Kimmelman & White, 2017) ➡ interesting, examining both cell-autonomous and non-cell-autonomous effects. This field is also seeing a shift in perception, recognising that autophagy may be tumour-suppressive at the early stages of tumour initiation, but promote tumour growth at later stages (possibly due to systemic interactions of the cancer with non-cancerous tissues).

A review of “Metabolic Flexibility in Health and Disease” (Goodpaster & Sparks, 2017) that will interest quite a few people as it examines fasting/refeeding paradigms and emphasises the role of skeletal muscle and adipose tissue. It also briefly examines evidence for and against the importance of individual tissues for the development of insulin resistance.

What I have read of interesting papers this week

Some useful reviews

One in Nature Medicine “Refining strategies to translate genome editing to the clinic” (Cornu et al. 2017). It covers some of the recent gene editing clinical trials and outlines key standardisations required to translate CRISPR and similar techniques into effective therapeutic strategies.

Brendand Manning and Alex Toker have written a comprehensive Cell review “AKT/PKB Signaling: Navigating the Network”. Looking forward to giving it a proper read!

Insulin signalling

Key paper in Cell: The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover (Tawo et al. 2017)

Apparently, previous studies have shown that although CHIP-null mice exhibit normal embryonic development, lack of CHIP (ubiquitin ligase, key for cellular protein quality control) accelerates ageing. Also, the livers of these mice develop insulin resistance. Using C. elegans and D. melanogaster, this study demonstrates that under non-stress conditions, CHIP ubiquitylates INSR and targets it for endocytic (not proteasomal) degradation; however, under proteotoxic stress, CHIP is “busy” sorting out other damaged proteins and this results in increased INSR levels and enhanced downstream activation of PI3K/AKT signalling. Interestingly, they make a link to severe INSR due to mutations in the receptor by mentioning K1095E; apparently, they have identified this residue to be ubiquitylated. So the findings are extrapolated to speculations about the effects of metabolic insults on INSR degradation and therefore glucose homeostasis.

Metabolic

Chad Cowan and co. on “Activation of IRF1 in Human Adipocytes Leads to Phenotypes Associated with Metabolic Disease” (Friesen et al. 2017, Stem Cell Reports)

From Jens Brüning’s lab on IL-6: IL-6 improves energy and glucose homeostasis in obesity via enhanced central IL-6 trans-signaling (Timper et al. 2017 Cell Reports) – not read in detail, but abstract suggests that similar to leptin, central IL-6 signalling suppresses feeding and improves glucose tolerance, but its actions are actually enhanced upon challenging mice with HFD. And the mechanism of signalling is interesting – the soluble IL-6R complexes with gp130 on the target cell surface, hence why they call this trans-signalling.

Obesity-induced hepatic steatosis is mediated by endoplasmic reticulum stress in the subfornical organ of the brain (Horwath et al. 2017, JCI Insight) – not read beyond abstract, but might be of relevance. Apparently reporting an uncoupling of hepatic steatosis from other obesity-linked conditions – i.e. reducing brain stress can reverse NAFLD, but without altering body weight, food intake, adiposity, or hypertension.

β-Klotho deficiency protects against obesity through a crosstalk between liver, microbiota, and brown adipose tissue (Somm et al. JCI Insight) – also only given this one a brief skim of the abstract and the introduction.

Some “old” stem cell signalling knowledge

An 10-year-old paper, but nonetheless interesting for me…

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling (Wang et al. 2007, Blood)

The abstract of this paper rightly starts out by stating that little is known about the cell-surface receptors (and implicit signalling pathways) that are activated under conditions that support stem cell self-renewal – despite improvements in developing defined conditions. Advances have been made in the past 10 years, but I would still argue that a lot remains to be learned.

The hESCs used in this study are maintained in MEF-conditioned medium on Matrigel or in defined medium supplemented with Heregulin-1B, Activin A, IGF1 & FGF2. Following various stimulations, the cell lysates are profiled with RTK arrays (42 human RTKs) to identify activated receptors. Hits are subsequently validated with inhibitor studies.

There is a strong phosphorylation of IGF1R and IR upon addition of conditioned medium, and the study then goes on to determine that it is IGF1R specifically that maintains self-renewal and prevents differentiation in stem cell maintenance conditions. Interestingly, they find that the stem cells express high levels of IGF1R (flow cytometry) and very low expression of insulin receptor (IR), which is consistent with my observations of absence of a response to insulin stimulation, but a clear activation of PI3K/AKT signalling in response to IGF1 stimulation. Looks quite solid and they also show a requirement for ERBB2/3 signalling. One thing that surprises me, though, is that they perform GF-depletion studies (DMEM/F12 + 0.5 % BSA) overnight which is really harsh for stem cells – mine start to day after 3h.

Another study was published in Nature at the same time as this paper, supporting the notion that IGF1R signalling in stem cells is essential for their self-renewal potential (Bendall et al. 2007, Nature).

Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of StemCell Differentiation (Petruk et al. 2017a, Molecular Cell) – there is an accompanying paper by the same group Petruk et al. 2017b in Cell Reports as well.

This is quite cool, by the group who originally developed a chromatin assembly assay relying on PLA (proximity ligation assay) to study the structure of nascent chromatin. Here, they show that immediately following DNA replication in stem cells, there is a delay in H3K27me3 deposition on the DNA, which allows transcription factors induced by differentiation signals to bind to their target sites. This is a very finely tuned time window that allows for exquisite control of differentiation only when the right transcription factors are induced and when the chromatin is open. Because the time window is limited, it also prevents spurious binding of other transcription factors later on, essentially allowing the cell to reconfigure itself in the process of differentiation once the primary transcription factors have done their job. There is an accompanying news and views as well: A Determined “Hesitation” on H3K27me3 Empowers Stem Cells to Differentiate (Huang and Wang 2017, Molecular Cell). In the Cell Reports paper, which I haven’t read, they apparently show that the same is true for HSCs.

Of general interest

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity (Saleheen et al. 2017, Nature)

Moving beyond non-human models to understand the complete  loss of certain genes in human. This is possible due to large scale sequencing of inbred populations that are likely to harbour two copies of rare mutations that result in complete loss of function for a particular gene. Really interesting and a more comprehensive catalogue of possible human knockouts is soon to come out. In this study, the authors link the discovered gene knockouts to more than 200 phenotypic traits, providing a detailed functional analysis. Interesting to go back and link the phenotypes to those observed in mice engineered to lack some of the same genes – you might be surprised at some of the discrepancies, cautioning us against thinking of mice as mini-humans. An accompanying news and views is also available at Nature.

 

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency (Yang et al. 2017, Cell)

This is quite ground-breaking in the developmental world (if it stands the test of independent replication). The study reports the first ever derivation of pluripotent stem cells (both mouse and human) with both extraembryonic (i.e. can generate placenta etc.) and embryonic capacity, terms extended pluripotent stem (EPS) cells.

Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation (Slobodin et al. 2017, Cell)

Quite cool both on the biological and techy side of things. Very, very nice approach to screen for differential promoter effects by using a barcoded reporter, essentially designing a new technique “Barcoded polysomal profiling” (BPP). Biologically, the study reports that mA6 of RNA is an epigenetic mechanism that determines the future activity and fate of the transcribed mRNA, thereby indirectly coupling transcription and translation.

Not read in detail, but this sounds a bit cool (apparently, you can change the sex of C. elegans with fatty acids): Fatty Acids Regulate Germline Sex Determination through ACS-4-Dependent Myristoylation (Tang et al. Cell 2017 quite impressive with only 2 authors!).

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a (Swarts et al. 2017, Molecular Cell –  from the Jinek lab) – just one of those papers that are good to have; Cas12a = Cpf1, the cousin of Cas9, also used for gene editing. Different from Cas9 by being able to process its own guide RNAs + different PAM requirement + Cpf1 generates staggered cuts.