bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–03–19
24 papers selected by
Ralitsa Radostinova Madsen, University College London
  1. PI3K-alpha translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in colorectal cancer.
  2. Identification of circular dorsal ruffles as signal platforms for the AKT pathway in glomerular podocytes.
  3. Regulation of mTORC1 by the Rag GTPases.
  4. Combined PTEN knockdown and local insulin in chronic experimental diabetic neuropathy.
  5. A general method for quantitative fractionation of mammalian cells.
  6. Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.
  7. De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics.
  8. Multi-range ERK responses shape the proliferative trajectory of single cells following oncogene induction.
  9. Preclinical evaluation of Insulin-like growth factor receptor 1 (IGF1R) and Insulin Receptor (IR) as a therapeutic targets in triple negative breast cancer.
  10. High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display.
  11. Precise homology-directed installation of large genomic edits in human cells with cleaving and nicking high-specificity Cas9 variants.
  12. Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.
  13. Passing the βAR: PI3Kγ Is the Judge of β Adrenergic Receptor Resensitization.
  14. Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.
  15. Biologically informed NeuralODEs for genome-wide regulatory dynamics.
  16. Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons.
  17. Lactate regulates cell cycle by remodeling the anaphase promoting complex.
  18. An optimized reporter of the transcription factor hypoxia-inducible factor 1α reveals complex HIF-1α activation dynamics in single cells.
  19. Turnover and replication analysis by isotope labeling (TRAIL) reveals the influence of tissue context on protein and organelle lifetimes.
  20. Spatial epigenome-transcriptome co-profiling of mammalian tissues.
  21. Reference compounds for characterizing cellular injury in high-content cellular morphology assays.
  22. A novel network approach to multiscale biological regulation.
  23. Medium depth influences O 2 availability and metabolism in cultured RPE cells.
  24. Screening in serum-derived medium reveals differential response to compounds targeting metabolism.
  1. Mol Cell Proteomics. 2023 Mar 15. pii: S1535-9476(23)00039-7. [Epub ahead of print] 100529
    PI3K-alpha translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in colorectal cancer.
    Michelle Palmieri, Bruno Catimel, Dmitri Mouradov, Anuratha Sakthianandeswaren, Eugene Kapp, Ching-Seng Ang, Nicholas A Williamson, Cameron J Nowell, Michael Christie, Jayesh Desai, Peter Gibbs, Antony W Burgess, Oliver M Sieber.
      The canonical view of phosphatidylinositol 3-kinase alpha (PI3Kα) signaling describes PtdIns(3,4,5)P3 generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin β-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by non-canonical PtdIns(3,4,5)P3 binding domains and showed overrepresentation for nuclear membrane, nucleolus and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of EGF-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.
    Keywords:  PtdIns(3,4,5)P(3) interactome; colorectal cancer; nuclear import pathway; phosphatidylinositol 3-kinase alpha; pre-mRNA splicing
    DOI:  https://doi.org/10.1016/j.mcpro.2023.100529
  2. J Cell Physiol. 2023 Mar 16.
    Identification of circular dorsal ruffles as signal platforms for the AKT pathway in glomerular podocytes.
    Rui Hua, Jinzi Wei, Mauricio Torres, Yuxin He, Yanan Li, Xiaowei Sun, Li Wang, Ken Inoki, Sei Yoshida.
      Circular dorsal ruffles (CDRs) are rounded membrane ruffles induced by growth factors to function as precursors of the large-scale endocytosis called macropinocytosis. In addition to their role in cellular uptake, recent research using cell line systems has shown that CDRs/macropinocytosis regulate the canonical AKT-mTORC1 growth factor signaling pathway. However, as CDRs have not been observed in tissues, their physiological relevance has remained unclear. Here, utilizing ultrahigh-resolution scanning electron microscopy, we first report that CDRs are expressed in glomerular podocytes ex vivo and in vivo, and we visually captured the transformation process to macropinocytosis. Moreover, through biochemical and imaging analyses, we show that AKT phosphorylation localized to CDRs upstream of mTORC1 activation in podocyte cell lines and isolated glomeruli. These results demonstrate the physiological role of CDRs as signal platforms for the AKT-mTORC1 pathway in glomerular podocytes at the tissue level. As mTORC1 plays critical roles in podocyte metabolism, and aberrant activation of mTORC1 triggers podocytopathies, our results strongly suggest that targeting CDR formation could represent a potential therapeutic approach for these diseases.
    Keywords:  AKT; circular dorsal ruffle; mTORC2; macropinocytosis; podocyte
    DOI:  https://doi.org/10.1002/jcp.30996
  3. Biochem Soc Trans. 2023 Mar 16. pii: BST20210038. [Epub ahead of print]
    Regulation of mTORC1 by the Rag GTPases.
    Tshering D Lama-Sherpa, Mi-Hyeon Jeong, Jenna L Jewell.
      The Rag GTPases are an evolutionarily conserved family that play a crucial role in amino acid sensing by the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is often referred to as the master regulator of cell growth. mTORC1 hyperactivation is observed in multiple diseases such as cancer, obesity, metabolic disorders, and neurodegeneration. The Rag GTPases sense amino acid levels and form heterodimers, where RagA or RagB binds to RagC or RagD, to recruit mTORC1 to the lysosome where it becomes activated. Here, we review amino acid signaling to mTORC1 through the Rag GTPases.
    Keywords:  Rag GTPases; amino acid sensing; mTORC1; signaling
    DOI:  https://doi.org/10.1042/BST20210038
  4. Diabetes. 2023 Mar 17. pii: db220743. [Epub ahead of print]
    Combined PTEN knockdown and local insulin in chronic experimental diabetic neuropathy.
    Vuong M Pham, Prashanth Komirishetty, Aparna Areti, Trevor Poitras, Nitish Thakor, Douglas W Zochodne.
      Diabetic polyneuropathy (DPN) renders progressive sensory neurodegeneration linked to hyperglycemia and its associated metabolopathy. Here we hypothesized that there may be additive impacts of direct insulin signaling, independent of glycemia and PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown on neuropathy. Our targets for combined interventions were neurons and Schwann cells in vitro and chronic Type 1 DPN in mice. IR (insulin receptor) expression was not altered by high glucose conditions in neurons or SCs and insulin promoted survival of neurons and proliferation of SCs in vitro. There were additive impacts between insulin signaling and PTEN knockdown in sensory neuron outgrowth and in axon myelination by SCs. In a chronic long term mouse model of experimental DPN unilateral intra-hindpaw injections of a PTEN siRNA and local insulin had additive impacts on correcting key features of chronic experimental DPN independent of glycemia, including motor axon conduction, thermal and mechanical sensory loss. Moreover, combined interventions improved sural and tibial nerve myelin thickness, hindpaw epidermal innervation and pAkt expression in dorsal root ganglia sensory neurons. We conclude that local PTEN inhibition or knockdown and insulin provide additive trophic support for sensory neurons and SCs whilst reversing key abnormalities of experimental DPN but without requiring metabolic correction.
    DOI:  https://doi.org/10.2337/db22-0743
  5. J Cell Biol. 2023 Jun 05. pii: e202209062. [Epub ahead of print]222(6):
    A general method for quantitative fractionation of mammalian cells.
    Yael Udi, Wenzhu Zhang, Milana E Stein, Inna Ricardo-Lax, Hilda A Pasolli, Brian T Chait, Michael P Rout.
      Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes.
    DOI:  https://doi.org/10.1083/jcb.202209062
  6. EMBO J. 2023 Mar 13. e112590
    Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.
    Lin Grimm, Elizabeth Mason, Hujun Yu, Stefanie Dudczig, Virginia Panara, Tyrone Chen, Neil I Bower, Scott Paterson, Maria Rondon Galeano, Sakurako Kobayashi, Anne Senabouth, Anne K Lagendijk, Joseph Powell, Kelly A Smith, Kazuhide S Okuda, Katarzyna Koltowska, Benjamin M Hogan.
      During development, the lymphatic vasculature forms as a second network derived chiefly from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is a key step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet the downstream mechanisms remain to be elucidated. We here present a single-cell transcriptomic atlas of lymphangiogenesis in zebrafish, revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single-cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a LEC-VEC fate shift. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new downstream targets and regulatory regions of the genome controlled by Prox1 and presents evidence that Prox1 specifies LEC fate primarily by limiting blood vascular and haematopoietic fate. This extensive single-cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.
    Keywords:  Notch1; Prox1; Vegfc single-cell sequencing; lymphangiogenesis; lymphatics
    DOI:  https://doi.org/10.15252/embj.2022112590
  7. Nat Commun. 2023 Mar 13. 14(1): 1362
    De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics.
    Leslie A Rowland, Adilson Guilherme, Felipe Henriques, Chloe DiMarzio, Sean Munroe, Nicole Wetoska, Mark Kelly, Keith Reddig, Gregory Hendricks, Meixia Pan, Xianlin Han, Olga R Ilkayeva, Christopher B Newgard, Michael P Czech.
      Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.
    DOI:  https://doi.org/10.1038/s41467-023-37016-8
  8. Cell Rep. 2023 Mar 14. pii: S2211-1247(23)00263-2. [Epub ahead of print]42(3): 112252
    Multi-range ERK responses shape the proliferative trajectory of single cells following oncogene induction.
    Jia-Yun Chen, Clemens Hug, José Reyes, Chengzhe Tian, Luca Gerosa, Fabian Fröhlich, Bas Ponsioen, Hugo J G Snippert, Sabrina L Spencer, Ashwini Jambhekar, Peter K Sorger, Galit Lahav.
      Oncogene-induced senescence is a phenomenon in which aberrant oncogene expression causes non-transformed cells to enter a non-proliferative state. Cells undergoing oncogenic induction display phenotypic heterogeneity, with some cells senescing and others remaining proliferative. The causes of heterogeneity remain unclear. We studied the sources of heterogeneity in the responses of human epithelial cells to oncogenic BRAFV600E expression. We found that a narrow expression range of BRAFV600E generated a wide range of activities of its downstream effector ERK. In population-level and single-cell assays, ERK activity displayed a non-monotonic relationship to proliferation, with intermediate ERK activities leading to maximal proliferation. We profiled gene expression across a range of ERK activities over time and characterized four distinct ERK response classes, which we propose act in concert to generate the ERK-proliferation response. Altogether, our studies map the input-output relationships between ERK activity and proliferation, elucidating how heterogeneity can be generated during oncogene induction.
    Keywords:  BRAF(V600E); CP: Cancer; ERK; cell cycle; heterogeneity; non-monotonic; oncogene-induced senescence; proliferation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112252
  9. PLoS One. 2023 ;18(3): e0282512
    Preclinical evaluation of Insulin-like growth factor receptor 1 (IGF1R) and Insulin Receptor (IR) as a therapeutic targets in triple negative breast cancer.
    Sandra Roche, Patricia Gaule, Deirdre Winrow, Nupur Mukherjee, Fiona O'Neill, Neil T Conlon, Justine Meiller, Denis M Collins, Alexandra Canonici, Mohammed Ibrahim Fawsi, Alejandra Estepa-Fernández, Stephen F Madden, John Crown, Norma O'Donovan, Alex J Eustace.
      Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, has fewer successful therapeutic therapies than other types of breast cancer. Insulin-like growth factor receptor 1 (IGF1R) and the Insulin receptor (IR) are associated with poor outcomes in TNBC. Targeting IGF1R has failed clinically. We aimed to test if inhibiting both IR/IGF1R was a rationale therapeutic approach to treat TNBC. We showed that despite IGF1R and IR being expressed in TNBC, their expression is not associated with a negative survival outcome. Furthermore, targeting both IR/IGF1R with inhibitors in multiple TNBC cell lines did not inhibit cell growth. Linsitinib, a small molecule inhibitor of both IGF1R and IR, did not block tumour formation and had no effect on tumour growth in vivo. Cumulatively these data suggest that while IGF1R and IR are expressed in TNBC, they are not good therapeutic targets. A potential reason for the limited anti-cancer impact when IR/IGF1R was targeted may be because multiple signalling pathways are altered in TNBC. Therefore, targeting individual signalling pathways may not be sufficient to inhibit cancer growth.
    DOI:  https://doi.org/10.1371/journal.pone.0282512
  10. Elife. 2023 Mar 16. pii: e82345. [Epub ahead of print]12
    High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display.
    Allyson Li, Rashmi Voleti, Minhee Lee, Dejan Gagoski, Neel H Shah.
      Tyrosine kinases and SH2 (phosphotyrosine recognition) domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue. Although the preferred recognition motifs of many kinases and SH2 domains are known, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically-encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases and SH2 domains. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences. We also screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. We extended this platform to the analysis of SH2 domains and showed that screens could predict relative binding affinities. Finally, we expanded our method to assess the impact of non-canonical and post-translationally modified amino acids on sequence recognition. This specificity profiling platform will shed new light on phosphotyrosine signaling and could readily be adapted to other protein modification/recognition domains.
    Keywords:  E. coli; biochemistry; chemical biology
    DOI:  https://doi.org/10.7554/eLife.82345
  11. Nucleic Acids Res. 2023 Mar 17. pii: gkad165. [Epub ahead of print]
    Precise homology-directed installation of large genomic edits in human cells with cleaving and nicking high-specificity Cas9 variants.
    Qian Wang, Jin Liu, Josephine M Janssen, Manuel A F V Gonçalves.
      Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.
    DOI:  https://doi.org/10.1093/nar/gkad165
  12. Cell Rep. 2023 Mar 10. pii: S2211-1247(23)00240-1. [Epub ahead of print]42(3): 112229
    Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.
    Aya O Satoh, Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Asuka Nanbo, Maho Amano, Yusuke Ohba.
      Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.
    Keywords:  CP: Cell biology; endocytosis; endosomes; imaging; interorganelle association; membrane association site; mitochondria; optogenetics; phosphoinositide 3-kinase (PI3K); voltage-dependent anion channel 2 (VDAC2)
    DOI:  https://doi.org/10.1016/j.celrep.2023.112229
  13. Circ Res. 2023 Mar 17. 132(6): 704-706
    Passing the βAR: PI3Kγ Is the Judge of β Adrenergic Receptor Resensitization.
    Justin M Quiles, Åsa B Gustafsson.
      
    Keywords:  beta-adrenergic receptor; endosomes; hypoxia; phosphoinositide 3 kinases
    DOI:  https://doi.org/10.1161/CIRCRESAHA.123.322554
  14. Science. 2023 Mar 16. eade3925
    Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.
    Jana Zecha, Florian P Bayer, Svenja Wiechmann, Julia Woortman, Nicola Berner, Julian Müller, Annika Schneider, Karl Kramer, Mar Abril-Gil, Thomas Hopf, Leonie Reichart, Lin Chen, Fynn M Hansen, Severin Lechner, Patroklos Samaras, Stephan Eckert, Ludwig Lautenbacher, Maria Reinecke, Firas Hamood, Polina Prokofeva, Larsen Vornholz, Chiara Falcomatà, Madeleine Dorsch, Ayla Schröder, Anton Venhuizen, Stephanie Wilhelm, Guillaume Médard, Gabriele Stoehr, Jürgen Ruland, Barbara M Grüner, Dieter Saur, Maike Buchner, Benjamin Ruprecht, Hannes Hahne, Matthew The, Mathias Wilhelm, Bernhard Kuster.
      Although most cancer drugs modulate the activities of cellular pathways by changing post-translational modifications (PTMs), surprisingly little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. Here, we introduce a proteomic assay termed decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action (MoA). Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B-cells by over-activating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.
    DOI:  https://doi.org/10.1126/science.ade3925
  15. bioRxiv. 2023 Feb 27. pii: 2023.02.24.529835. [Epub ahead of print]
    Biologically informed NeuralODEs for genome-wide regulatory dynamics.
    Intekhab Hossain, Viola Fanfani, John Quackenbush, Rebekka Burkholz.
      Models that are formulated as ordinary differential equations (ODEs) can accurately explain temporal gene expression patterns and promise to yield new insights into important cellular processes, disease progression, and intervention design. Learning such ODEs is challenging, since we want to predict the evolution of gene expression in a way that accurately encodes the causal gene-regulatory network (GRN) governing the dynamics and the nonlinear functional relationships between genes. Most widely used ODE estimation methods either impose too many parametric restrictions or are not guided by meaningful biological insights, both of which impedes scalability and/or explainability. To overcome these limitations, we developed PHOENIX, a modeling framework based on neural ordinary differential equations (NeuralODEs) and Hill-Langmuir kinetics, that can flexibly incorporate prior domain knowledge and biological constraints to promote sparse, biologically interpretable representations of ODEs. We test accuracy of PHOENIX in a series of in silico experiments benchmarking it against several currently used tools for ODE estimation. We also demonstrate PHOENIX’s flexibility by studying oscillating expression data from synchronized yeast cells and assess its scalability by modelling genome-scale breast cancer expression for samples ordered in pseudotime. Finally, we show how the combination of user-defined prior knowledge and functional forms from systems biology allows PHOENIX to encode key properties of the underlying GRN, and subsequently predict expression patterns in a biologically explainable way. PHOENIX will be available as open source at: https://github.com/QuackenbushLab/phoenix .
    DOI:  https://doi.org/10.1101/2023.02.24.529835
  16. Mol Cell. 2023 Mar 03. pii: S1097-2765(23)00114-4. [Epub ahead of print]
    Massively parallel characterization of CRISPR activator efficacy in human induced pluripotent stem cells and neurons.
    Qianxin Wu, Junjing Wu, Kaiser Karim, Xi Chen, Tengyao Wang, Sho Iwama, Stefania Carobbio, Peter Keen, Antonio Vidal-Puig, Mark R Kotter, Andrew Bassett.
      CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.
    Keywords:  CRISPR activation; CRISPRa; VPR; chromatin; epigenetic; hiPSC; iNeurons; p300; single cell; stem cells
    DOI:  https://doi.org/10.1016/j.molcel.2023.02.011
  17. Nature. 2023 Mar 15.
    Lactate regulates cell cycle by remodeling the anaphase promoting complex.
    Weihai Liu, Yun Wang, Luiz H M Bozi, Patrick Fischer, Mark P Jedrychowski, Haopeng Xiao, Tao Wu, Narek Darabedian, Xiadi He, Evanna L Mills, Nils Burger, Sanghee Shin, Anita Reddy, Hans-Georg Sprenger, Nhien Tran, Sally Winther, Stephen M Hinshaw, Jingnan Shen, Hyuk-Soo Seo, Kijun Song, Andrew Z Xu, Luke Sebastian, Jean Zhao, Sirano Dhe-Paganon, Jianwei Che, Steven P Gygi, Haribabu Arthanari, Edward T Chouchani.
      Lactate is abundant in rapidly dividing cells due to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here, we deploy a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we elucidate a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodeling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We discover that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. The above mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient replete growth phase to stimulate timed opening of APC/C, cell division, and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodeling and can overcome anti-mitotic pharmacology via mitotic slippage. Taken together, we define a biochemical mechanism through which lactate directly regulates protein function to control cell cycle and proliferation.
    DOI:  https://doi.org/10.1038/s41586-023-05939-3
  18. J Biol Chem. 2023 Mar 10. pii: S0021-9258(23)00241-7. [Epub ahead of print] 104599
    An optimized reporter of the transcription factor hypoxia-inducible factor 1α reveals complex HIF-1α activation dynamics in single cells.
    Stevan Jeknić, Takamasa Kudo, Joanna J Song, Markus W Covert.
      Immune cells adopt a variety of metabolic states to support their many biological functions, which include fighting pathogens, removing tissue debris, and tissue remodeling. One of the key mediators of these metabolic changes is the transcription factor hypoxia-inducible factor 1α (HIF-1α). Single-cell dynamics have been shown to be an important determinant of cell behavior; however, despite the importance of HIF-1α, little is known about its single-cell dynamics or their effect on metabolism. To address this knowledge gap, here we optimized a HIF-1α fluorescent reporter and applied it to study single-cell dynamics. First, we showed that single cells are likely able to differentiate multiple levels of prolyl hydroxylase inhibition, a marker of metabolic change, via HIF-1α activity. We then applied a physiological stimulus known to trigger metabolic change, interferon-γ, and observed heterogeneous, oscillatory HIF-1α responses in single cells. Finally, we input these dynamics into a mathematical model of HIF-1α-regulated metabolism, and discovered a profound difference between cells exhibiting high versus low HIF-1α activation. Specifically, we found cells with high HIF-1α activation are able to meaningfully reduce flux through the tricarboxylic acid cycle and show a notable increase in the NAD+/NADH ratio compared to cells displaying low HIF-1α activation. Altogether, this work demonstrates an optimized reporter for studying HIF-1α in single cells and reveals previously unknown principles of HIF-1α activation.
    Keywords:  fluorescence; hypoxia-inducible factor (HIF); mathematical modeling; microscopy; systems biology
    DOI:  https://doi.org/10.1016/j.jbc.2023.104599
  19. Mol Syst Biol. 2023 Mar 17. e11393
    Turnover and replication analysis by isotope labeling (TRAIL) reveals the influence of tissue context on protein and organelle lifetimes.
    John Hasper, Kevin Welle, Jennifer Hryhorenko, Sina Ghaemmaghami, Abigail Buchwalter.
      The lifespans of proteins range from minutes to years within mammalian tissues. Protein lifespan is relevant to organismal aging, as long-lived proteins accrue damage over time. It is unclear how protein lifetime is shaped by tissue context, where both cell turnover and proteolytic degradation contribute to protein turnover. We develop turnover and replication analysis by 15 N isotope labeling (TRAIL) to quantify protein and cell lifetimes with high precision and demonstrate that cell turnover, sequence-encoded features, and environmental factors modulate protein lifespan across tissues. Cell and protein turnover flux are comparable in proliferative tissues, while protein turnover outpaces cell turnover in slowly proliferative tissues. Physicochemical features such as hydrophobicity, charge, and disorder influence protein turnover in slowly proliferative tissues, but protein turnover is much less sequence-selective in highly proliferative tissues. Protein lifetimes vary nonrandomly across tissues after correcting for cell turnover. Multiprotein complexes such as the ribosome have consistent lifetimes across tissues, while mitochondria, peroxisomes, and lipid droplets have variable lifetimes. TRAIL can be used to explore how environment, aging, and disease affect tissue homeostasis.
    Keywords:  metabolic labeling; protein lifetime; proteostasis; tissue homeostasis; tool development
    DOI:  https://doi.org/10.15252/msb.202211393
  20. Nature. 2023 Mar 15.
    Spatial epigenome-transcriptome co-profiling of mammalian tissues.
    Di Zhang, Yanxiang Deng, Petra Kukanja, Eneritz Agirre, Marek Bartosovic, Mingze Dong, Cong Ma, Sai Ma, Graham Su, Shuozhen Bao, Yang Liu, Yang Xiao, Gorazd B Rosoklija, Andrew J Dwork, J John Mann, Kam W Leong, Maura Boldrini, Liya Wang, Maximilian Haeussler, Benjamin J Raphael, Yuval Kluger, Gonçalo Castelo-Branco, Rong Fan.
      Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context1-5. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.
    DOI:  https://doi.org/10.1038/s41586-023-05795-1
  21. Nat Commun. 2023 Mar 13. 14(1): 1364
    Reference compounds for characterizing cellular injury in high-content cellular morphology assays.
    Jayme L Dahlin, Bruce K Hua, Beth E Zucconi, Shawn D Nelson, Shantanu Singh, Anne E Carpenter, Jonathan H Shrimp, Evelyne Lima-Fernandes, Mathias J Wawer, Lawrence P W Chung, Ayushi Agrawal, Mary O'Reilly, Dalia Barsyte-Lovejoy, Magdalena Szewczyk, Fengling Li, Parnian Lak, Matthew Cuellar, Philip A Cole, Jordan L Meier, Tim Thomas, Jonathan B Baell, Peter J Brown, Michael A Walters, Paul A Clemons, Stuart L Schreiber, Bridget K Wagner.
      Robust, generalizable approaches to identify compounds efficiently with undesirable mechanisms of action in complex cellular assays remain elusive. Such a process would be useful for hit triage during high-throughput screening and, ultimately, predictive toxicology during drug development. Here we generate cell painting and cellular health profiles for 218 prototypical cytotoxic and nuisance compounds in U-2 OS cells in a concentration-response format. A diversity of compounds that cause cellular damage produces bioactive cell painting morphologies, including cytoskeletal poisons, genotoxins, nonspecific electrophiles, and redox-active compounds. Further, we show that lower quality lysine acetyltransferase inhibitors and nonspecific electrophiles can be distinguished from more selective counterparts. We propose that the purposeful inclusion of cytotoxic and nuisance reference compounds such as those profiled in this resource will help with assay optimization and compound prioritization in complex cellular assays like cell painting.
    DOI:  https://doi.org/10.1038/s41467-023-36829-x
  22. Cell Syst. 2023 Mar 15. pii: S2405-4712(23)00056-X. [Epub ahead of print]14(3): 177-179
    A novel network approach to multiscale biological regulation.
    Guido Gigante, Alessandro Giuliani, Maurizio Mattia.
      Modeling systems at multiple interacting scales is probably the most relevant task for pursuing a physically motivated explanation of biological regulation. In a new study, Smart and Zilman develop a convincing, albeit preliminary, model of the interplay between the cell microscale and the macroscopic tissue organization in biological systems.
    DOI:  https://doi.org/10.1016/j.cels.2023.02.004
  23. bioRxiv. 2023 Mar 02. pii: 2023.03.01.530623. [Epub ahead of print]
    Medium depth influences O 2 availability and metabolism in cultured RPE cells.
    Daniel T Hass, Qitao Zhang, Gillian Autterson, Richard Bryan, James B Hurley, Jason Ml Miller.
       Purpose: RPE oxidative metabolism is critical for normal retinal function and is often studied in cell culture systems. Here, we show that conventional culture media volumes dramatically impact O 2 availability, limiting oxidative metabolism. We suggest optimal conditions to ensure cultured RPE is in a normoxic environment permissive to oxidative metabolism.
    Methods: We altered the availability of O 2 to human primary RPE cultures directly via a hypoxia chamber or indirectly via the amount of medium over cells. We measured oxygen consumption rates (OCR), glucose consumption, lactate production, 13 C 6 -glucose flux, hypoxia inducible factor (HIF-1α) stability, intracellular lipid droplets after a lipid challenge, trans-epithelial electrical resistance, cell morphology, and pigmentation.
    Results: Medium volumes commonly employed during RPE culture limit diffusion of O 2 to cells, triggering hypoxia, activating HIF-1α, limiting OCR, and dramatically altering cell metabolism, with only minor effects on typical markers of RPE health. Media volume effects on O 2 availability decrease acetyl-CoA utilization, increase glycolysis, and alter the size and number of intracellular lipid droplets under lipid-rich conditions.
    Conclusions: Despite having little impact on visible and typical markers of RPE culture health, media volume dramatically affects RPE physiology "under the hood". As RPE-centric diseases like age-related macular degeneration (AMD) involve oxidative metabolism, RPE cultures need to be optimized to study such diseases. We provide guidelines for optimal RPE culture volumes that balance ample nutrient availability from larger media volumes with adequate O 2 availability seen with smaller media volumes.
    DOI:  https://doi.org/10.1101/2023.03.01.530623
  24. bioRxiv. 2023 Feb 27. pii: 2023.02.25.529972. [Epub ahead of print]
    Screening in serum-derived medium reveals differential response to compounds targeting metabolism.
    Keene L Abbott, Ahmed Ali, Dominick Casalena, Brian T Do, Raphael Ferreira, Jaime H Cheah, Christian K Soule, Amy Deik, Tenzin Kunchok, Daniel R Schmidt, Steffen Renner, Sophie E Honeder, Michelle Wu, Sze Ham Chan, Tenzin Tseyang, Daniel Greaves, Peggy P Hsu, Christopher W Ng, Chelsea J Zhang, Ali Farsidjani, Iva Monique T Gramatikov, Nicholas J Matheson, Caroline A Lewis, Clary B Clish, Matthew G Rees, Jennifer A Roth, Lesley Mathews Griner, Alexander Muir, Douglas S Auld, Matthew G Vander Heiden.
      A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.
    DOI:  https://doi.org/10.1101/2023.02.25.529972
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