bims-pideca 2021-03-07 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2021–03–07
thirty-two papers selected by
Ralitsa Radostinova Madsen, University College London

iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation.
Pre-existing Cell States Control Heterogeneity of Both EGFR and CXCR4 Signaling.
Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.
The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals.
Signaling Heterogeneity is Defined by Pathway Architecture and Intercellular Variability in Protein Expression.
Fast searches of large collections of single-cell data using scfind.
Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism.
An expanded universe of cancer targets.
Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.
Dysregulation of myelin synthesis and actomyosin function underlies aberrant myelin in CMT4B1 neuropathy.
Novel Regulators of the IGF System in Cancer.
Defective insulin receptor signaling in hPSCs skews pluripotency and negatively perturbs neural differentiation.
Metabolic regulation of cell competition.
mTORC1-chaperonin CCT signaling regulates m6A RNA methylation to suppress autophagy.
A CRISPR Competition Assay to Identify Cancer Genetic Dependencies.
Multi-omics analysis reveals contextual tumor suppressive and oncogenic gene modules within the acute hypoxic response.
Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.
Hippo signalling maintains ER expression and ER+ breast cancer growth.
Long Noncoding RNAs in Human Stemness and Differentiation.
Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics.
Clinical experience with the AKT1 inhibitor miransertib in two children with PIK3CA-related overgrowth syndrome.
Molecular Biomarkers for Contemporary Therapies in Hormone Receptor-Positive Breast Cancer.
How does obesity promote breast cancer tumor growth?
Cancer therapies based on targeted protein degradation - lessons learned with lenalidomide.
Quantifying Cancer: More Than Just a Numbers Game.
Akt Is S-Palmitoylated: A New Layer of Regulation for Akt.
Challenges in Studying Stem Cell Metabolism.
Adipocyte Integrin-Linked Kinase Plays a Key Role in the Development of Diet-Induced Adipose Insulin Resistance in Male Mice.
Reduced insulin/IGF1 signaling prevents immune aging via ZIP-10/bZIP-mediated feedforward loop.
MYC inhibition, courtesy of E. coli.
Fine-tuning p53 activity by modulating the interaction between eukaryotic translation initiation factor eIF4E and RNA-binding protein RBM38.
CRISPR screens in physiologic medium reveal conditionally essential genes in human cells.

Front Oncol. 2021 ;11 631766

iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation.

Zhen Ding, Dai Ogata, Jason Roszik, Yong Qin, Sun-Hee Kim, Michael T Tetzlaff, Alexander J Lazar, Michael A Davies, Suhendan Ekmekcioglu, Elizabeth A Grimm.

  We previously showed that inducible nitric oxide synthase (iNOS) protein expression in melanoma tumor cells is associated with poor patient prognosis. Here, we analyzed the association between iNOS and the oncogenic PI3K-AKT pathway. TCGA data show that iNOS and phospho-Akt Ser473 expression were associated significantly only in the subset of tumors with genetically intact PTEN. Employing a stage III melanoma TMA, we showed that iNOS protein presence is significantly associated with shorter survival only in tumors with PTEN protein expression. These findings led to our hypothesis that the iNOS product, nitric oxide (NO), suppresses the function of PTEN and stimulates PI3K-Akt activation. Melanoma cells in response to NO exposure in vitro exhibited enhanced AKT kinase activity and substrate phosphorylation, as well as attenuated PTEN phosphatase activity. Biochemical analysis showed that NO exposure resulted in a post-translationally modified S-Nitrosylation (SNO) PTEN, which was also found in cells expressing iNOS. Our findings provide evidence that NO-rich cancers may exhibit AKT activation due to post-translational inactivation of PTEN. This unique activation of oncogenic pathway under nitrosative stress may contribute to the pathogenesis of iNOS in melanoma. Significance: Our study shows that iNOS expression is associated with increased PI3K-AKT signaling and worse clinical outcomes in melanoma patients with wt (intact) PTEN. Mutated PTEN is already inactivated. We also demonstrate that NO activates the PI3K-AKT pathway by suppressing PTEN suppressor function concurrent with the formation of PTEN-SNO. This discovery provides insight into the consequences of inflammatory NO produced in human melanoma and microenvironmental cells. It suggests that NO-driven modification provides a marker of PTEN inactivation, and represents a plausible mechanism of tumor suppressor inactivation in iNOS expressing subset of cancers.

Keywords:  AKT activation; PI3K-AKT axis; S-nitrosylation; inducible nitric oxide synthase; melanoma; nitric oxide; phosphatase and tensin homolog

DOI:  https://doi.org/10.3389/fonc.2021.631766
Cell Mol Bioeng. 2021 Feb;14(1): 49-64

Pre-existing Cell States Control Heterogeneity of Both EGFR and CXCR4 Signaling.

Phillip C Spinosa, Patrick C Kinnunen, Brock A Humphries, Gary D Luker, Kathryn E Luker, Jennifer J Linderman.

   Introduction: CXCR4 and epidermal growth factor receptor (EGFR) represent two major families of receptors, G-protein coupled receptors and receptor tyrosine kinases, with central functions in cancer. While utilizing different upstream signaling molecules, both CXCR4 and EGFR activate kinases ERK and Akt, although single-cell activation of these kinases is markedly heterogeneous. One hypothesis regarding the origin of signaling heterogeneity proposes that intercellular variations arise from differences in pre-existing intracellular states set by extrinsic noise. While pre-existing cell states vary among cells, each pre-existing state defines deterministic signaling outputs to downstream effectors. Understanding causes of signaling heterogeneity will inform treatment of cancers with drugs targeting drivers of oncogenic signaling.
Methods: We built a single-cell computational model to predict Akt and ERK responses to CXCR4- and EGFR-mediated stimulation. We investigated signaling heterogeneity through these receptors and tested model predictions using quantitative, live-cell time-lapse imaging.
Results: We show that the pre-existing cell state predicts single-cell signaling through both CXCR4 and EGFR. Computational modeling reveals that the same set of pre-existing cell states explains signaling heterogeneity through both EGFR and CXCR4 at multiple doses of ligands and in two different breast cancer cell lines. The model also predicts how phosphatidylinositol-3-kinase (PI3K) targeted therapies potentiate ERK signaling in certain breast cancer cells and that low level, combined inhibition of MEK and PI3K ablates potentiated ERK signaling.
Conclusions: Our data demonstrate that a conserved motif exists for EGFR and CXCR4 signaling and suggest potential clinical utility of the computational model to optimize therapy.

Keywords:  Computational modeling; Heterogeneity; Kinase; Molecular imaging; Single cell

DOI:  https://doi.org/10.1007/s12195-020-00640-1
Elife. 2021 Mar 04. pii: e64691. [Epub ahead of print]10

Disease related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Manoj K Rathinaswamy, Zied Gaieb, Kaelin D Fleming, Chiara Borsari, Noah J Harris, Brandon J Moeller, Matthias P Wymann, Rommie E Amaro, John E Burke.

  Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110g) playing key roles in immune signalling. p110g is a key factor in inflammatory diseases, and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall this work provides unique insights into regulatory mechanisms that control PI3Kg kinase activity, and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

Keywords:  biochemistry; chemical biology; human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.64691
Elife. 2021 Mar 01. pii: e63326. [Epub ahead of print]10

The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals.

Margaret E Torrence, Michael R MacArthur, Aaron M Hosios, Alexander J Valvezan, John M Asara, James R Mitchell, Brendan D Manning.

  The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast (MEF) and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.

Keywords:  cancer biology; cell biology; human; mouse; rat

DOI:  https://doi.org/10.7554/eLife.63326
iScience. 2021 Feb 19. 24(2): 102118

Signaling Heterogeneity is Defined by Pathway Architecture and Intercellular Variability in Protein Expression.

Dougall Norris, Pengyi Yang, Sung-Young Shin, Alison L Kearney, Hani Jieun Kim, Thomas Geddes, Alistair M Senior, Daniel J Fazakerley, Lan K Nguyen, David E James, James G Burchfield.

  Insulin's activation of PI3K/Akt signaling, stimulates glucose uptake by enhancing delivery of GLUT4 to the cell surface. Here we examined the origins of intercellular heterogeneity in insulin signaling. Akt activation alone accounted for ~25% of the variance in GLUT4, indicating that additional sources of variance exist. The Akt and GLUT4 responses were highly reproducible within the same cell, suggesting the variance is between cells (extrinsic) and not within cells (intrinsic). Generalized mechanistic models (supported by experimental observations) demonstrated that the correlation between the steady-state levels of two measured signaling processes decreases with increasing distance from each other and that intercellular variation in protein expression (as an example of extrinsic variance) is sufficient to account for the variance in and between Akt and GLUT4. Thus, the response of a population to insulin signaling is underpinned by considerable single-cell heterogeneity that is largely driven by variance in gene/protein expression between cells.

Keywords:  Cell Biology; Experimental Models in Systems Biology; Mathematical Biosciences; Systems Biology

DOI:  https://doi.org/10.1016/j.isci.2021.102118
Nat Methods. 2021 Mar 01.

Fast searches of large collections of single-cell data using scfind.

Jimmy Tsz Hang Lee, Nikolaos Patikas, Vladimir Yu Kiselev, Martin Hemberg.

Single-cell technologies have made it possible to profile millions of cells, but for these resources to be useful they must be easy to query and access. To facilitate interactive and intuitive access to single-cell data we have developed scfind, a single-cell analysis tool that facilitates fast search of biologically or clinically relevant marker genes in cell atlases. Using transcriptome data from six mouse cell atlases, we show how scfind can be used to evaluate marker genes, perform in silico gating, and identify both cell-type-specific and housekeeping genes. Moreover, we have developed a subquery optimization routine to ensure that long and complex queries return meaningful results. To make scfind more user friendly, we use indices of PubMed abstracts and techniques from natural language processing to allow for arbitrary queries. Finally, we show how scfind can be used for multi-omics analyses by combining single-cell ATAC-seq data with transcriptome data.

DOI: https://doi.org/10.1038/s41592-021-01076-9
EMBO Mol Med. 2021 Mar 05. e12461

Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism.

Goetz Hartleben, Kenji Schorpp, Yun Kwon, Barbara Betz, Foivos-Filippos Tsokanos, Zahra Dantes, Arlett Schäfer, Ina Rothenaigner, José Manuel Monroy Kuhn, Pauline Morigny, Lisa Mehr, Sean Lin, Susanne Seitz, Janina Tokarz, Anna Artati, Jerzy Adamsky, Oliver Plettenburg, Dominik Lutter, Martin Irmler, Johannes Beckers, Maximilian Reichert, Kamyar Hadian, Anja Zeigerer, Stephan Herzig, Mauricio Berriel Diaz.

  By accentuating drug efficacy and impeding resistance mechanisms, combinatorial, multi-agent therapies have emerged as key approaches in the treatment of complex diseases, most notably cancer. Using high-throughput drug screens, we uncovered distinct metabolic vulnerabilities and thereby identified drug combinations synergistically causing a starvation-like lethal catabolic response in tumor cells from different cancer entities. Domperidone, a dopamine receptor antagonist, as well as several tricyclic antidepressants (TCAs), including imipramine, induced cancer cell death in combination with the mitochondrial uncoupler niclosamide ethanolamine (NEN) through activation of the integrated stress response pathway and the catabolic CLEAR network. Using transcriptome and metabolome analyses, we characterized a combinatorial response, mainly driven by the transcription factors CHOP and TFE3, which resulted in cell death through enhanced pyrimidine catabolism as well as reduced pyrimidine synthesis. Remarkably, the drug combinations sensitized human organoid cultures to the standard-of-care chemotherapy paclitaxel. Thus, our combinatorial approach could be clinically implemented into established treatment regimen, which would be further facilitated by the advantages of drug repurposing.

Keywords:  cancer metabolism; integrated stress response; metabolic vulnerabilities; pyrimidine metabolism; tricyclic antidepressants

DOI:  https://doi.org/10.15252/emmm.202012461
Cell. 2021 Mar 04. pii: S0092-8674(21)00170-7. [Epub ahead of print]184(5): 1142-1155

An expanded universe of cancer targets.

Cancer Target Discovery and Development Network

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

DOI: https://doi.org/10.1016/j.cell.2021.02.020
Nat Commun. 2021 03 03. 12(1): 1407

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.

Lars Custers, Eleonora Khabirova, Tim H H Coorens, Thomas R W Oliver, Camilla Calandrini, Matthew D Young, Felipe A Vieira Braga, Peter Ellis, Lira Mamanova, Heidi Segers, Arie Maat, Marcel Kool, Eelco W Hoving, Marry M van den Heuvel-Eibrink, James Nicholson, Karin Straathof, Liz Hook, Ronald R de Krijger, Claire Trayers, Kieren Allinson, Sam Behjati, Jarno Drost.

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies.

DOI: https://doi.org/10.1038/s41467-021-21675-6
Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2009469118. [Epub ahead of print]118(10):

Dysregulation of myelin synthesis and actomyosin function underlies aberrant myelin in CMT4B1 neuropathy.

Marta Guerrero-Valero, Federica Grandi, Silvia Cipriani, Valeria Alberizzi, Roberta Di Guardo, Gaetan Chicanne, Linda Sawade, Francesca Bianchi, Ubaldo Del Carro, Ivan De Curtis, Davide Pareyson, Yesim Parman, Angelo Schenone, Volker Haucke, Bernard Payrastre, Alessandra Bolino.

  Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P 2, with a preference for PtdIns(3,5)P 2 A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P 2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P 2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth.

Keywords:  Charcot-Marie-Tooth neuropathies; Schwann cells; myelin; myotubularin; phosphoinositides

DOI:  https://doi.org/10.1073/pnas.2009469118
Biomolecules. 2021 Feb 12. pii: 273. [Epub ahead of print]11(2):

Novel Regulators of the IGF System in Cancer.

Caterina Mancarella, Andrea Morrione, Katia Scotlandi.

  The insulin-like growth factor (IGF) system is a dynamic network of proteins, which includes cognate ligands, membrane receptors, ligand binding proteins and functional downstream effectors. It plays a critical role in regulating several important physiological processes including cell growth, metabolism and differentiation. Importantly, alterations in expression levels or activation of components of the IGF network are implicated in many pathological conditions including diabetes, obesity and cancer initiation and progression. In this review we will initially cover some general aspects of IGF action and regulation in cancer and then focus in particular on the role of transcriptional regulators and novel interacting proteins, which functionally contribute in fine tuning IGF1R signaling in several cancer models. A deeper understanding of the biological relevance of this network of IGF1R modulators might provide novel therapeutic opportunities to block this system in neoplasia.

Keywords:  ADAR; DDR1; E-cadherin; IGF system; IGF2BPs; cancer; circular RNAs; decorin; functional regulation; transcriptional regulators

DOI:  https://doi.org/10.3390/biom11020273
J Biol Chem. 2021 Mar 02. pii: S0021-9258(21)00270-2. [Epub ahead of print] 100495

Defective insulin receptor signaling in hPSCs skews pluripotency and negatively perturbs neural differentiation.

Adrian Kee Keong Teo, Linh Nguyen, Manoj K Gupta, Hwee Hui Lau, Larry Sai Weng Loo, Nicholas Jackson, Chang Siang Lim, William Mallard, Marina A Gritsenko, John L Rinn, Richard D Smith, Wei-Jun Qian, Rohit N Kulkarni.

Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells (hPSCs) and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Importantly, RNA-sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry-based proteomic analyses further confirmed a global down-regulation of extracellular matrix (ECM) proteins. Subsequent differentiation towards the neural lineage reflected alterations in SOX1+PAX6+ neuroectoderm and FOXG1+ cortical neuron marker expression, and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the ECM necessary for the stem cell niche and regulating cell fate specification including the neural lineage.

DOI: https://doi.org/10.1016/j.jbc.2021.100495
Dev Biol. 2021 Feb 27. pii: S0012-1606(21)00051-8. [Epub ahead of print]

Metabolic regulation of cell competition.

Lorena Esteban-Martínez, Miguel Torres.

  Cell Competition is a selective process by which viable cells are eliminated from developing or adult tissues by interactions with their neighbors. In many cases, the eliminated cells (losers) display reduced fitness, yet they would be able to sustain tissue growth or maintenance in a homotypic environment, and are only eliminated when confronted with surrounding wild type cells (winners). In addition, cells with oncogenic mutations that do not show reduced fitness can also be eliminated from tissues when surrounded by wild type cells. Depending on the context, transformed cells can also become supercompetitors and eliminate surrounding wild type cells, thereby promoting tumor formation. Several factors have been shown to play essential roles in Cell Competition, including genes relevant in developmental growth, tumor formation and epithelial apico-basal polarity. Recent discoveries, however, suggest that energy metabolism plays a central role in very different models of cell competition. Here we review the involvement of mitochondrial dynamics and metabolism, autophagy and nutritional status in cell competition and discuss the possible implications of this emerging field.

Keywords:  Apoptosis; Autophagy; Energy metabolism; Glycolysis; Lactate; Mitochondria; Tumor formation; Tumor suppression; Warburg effect

DOI:  https://doi.org/10.1016/j.ydbio.2021.02.011
Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2021945118. [Epub ahead of print]118(10):

mTORC1-chaperonin CCT signaling regulates m6A RNA methylation to suppress autophagy.

Hong-Wen Tang, Jui-Hsia Weng, Wen Xing Lee, Yanhui Hu, Lei Gu, Sungyun Cho, Gina Lee, Richard Binari, Cathleen Li, Min En Cheng, Ah-Ram Kim, Jun Xu, Zhangfei Shen, Chiwei Xu, John M Asara, John Blenis, Norbert Perrimon.

  Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.

Keywords:  autophagy; chaperonin containing Tailless complex polypeptide 1 (CCT); m6A RNA methylation; m6A methyltransferase complex (MTC); mTORC1

DOI:  https://doi.org/10.1073/pnas.2021945118
Bio Protoc. 2020 Jul 20. 10(14): e3682

A CRISPR Competition Assay to Identify Cancer Genetic Dependencies.

Vishruth Girish, Jason M Sheltzer.

  The CRISPR/Cas9 system is a powerful tool for genome editing, wherein the RNA-guided nuclease Cas9 can be directed to introduce double-stranded breaks (DSBs) at a targeted locus. In mammalian cells, these DSBs are typically repaired through error-prone processes, resulting in insertions or deletions (indels) at the targeted locus. Researchers can use these Cas9-mediated lesions to probe the consequences of loss-of-function perturbations in genes of interest. Here, we describe an optimized protocol to identify specific genes required for cancer cell fitness through a CRISPR-mediated cellular competition assay. Identifying these genetic dependencies is of utmost importance, as they provide potential targets for anti-cancer drug development. This protocol provides researchers with a robust and scalable approach to investigate gene dependencies in a variety of cell lines and cancer types and to validate the results of high-throughput or whole-genome screens.

Keywords:  CRISPR; Cancer; Cell competition; Cell fitness; Essential genes; Genetic dependency

DOI:  https://doi.org/10.21769/BioProtoc.3682
Nat Commun. 2021 03 02. 12(1): 1375

Multi-omics analysis reveals contextual tumor suppressive and oncogenic gene modules within the acute hypoxic response.

Zdenek Andrysik, Heather Bender, Matthew D Galbraith, Joaquin M Espinosa.

Cellular adaptation to hypoxia is a hallmark of cancer, but the relative contribution of hypoxia-inducible factors (HIFs) versus other oxygen sensors to tumorigenesis is unclear. We employ a multi-omics pipeline including measurements of nascent RNA to characterize transcriptional changes upon acute hypoxia. We identify an immediate early transcriptional response that is strongly dependent on HIF1A and the kinase activity of its cofactor CDK8, includes indirect repression of MYC targets, and is highly conserved across cancer types. HIF1A drives this acute response via conserved high-occupancy enhancers. Genetic screen data indicates that, in normoxia, HIF1A displays strong cell-autonomous tumor suppressive effects through a gene module mediating mTOR inhibition. Conversely, in advanced malignancies, expression of a module of HIF1A targets involved in collagen remodeling is associated with poor prognosis across diverse cancer types. In this work, we provide a valuable resource for investigating context-dependent roles of HIF1A and its targets in cancer biology.

DOI: https://doi.org/10.1038/s41467-021-21687-2
Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2003014118. [Epub ahead of print]118(10):

Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.

Pei-Yun Tsai, Min-Sik Lee, Unmesh Jadhav, Insia Naqvi, Shariq Madha, Ashley Adler, Meeta Mistry, Sergey Naumenko, Caroline A Lewis, Daniel S Hitchcock, Frederick R Roberts, Peter DelNero, Thomas Hank, Kim C Honselmann, Vicente Morales Oyarvide, Mari Mino-Kenudson, Clary B Clish, Ramesh A Shivdasani, Nada Y Kalaany.

  Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.

Keywords:  epigenetics; glutamine synthetase; mTORC1; nutrient deprivation; pancreatic cancer

DOI:  https://doi.org/10.1073/pnas.2003014118
Nature. 2021 Mar;591(7848): E1-E10

Hippo signalling maintains ER expression and ER+ breast cancer growth.

Shenghong Ma, Zhengming Wu, Feng Yang, Jianmin Zhang, Randy L Johnson, Michael G Rosenfeld, Kun-Liang Guan.

DOI: https://doi.org/10.1038/s41586-020-03131-5
Trends Cell Biol. 2021 Mar 01. pii: S0962-8924(21)00027-1. [Epub ahead of print]

Long Noncoding RNAs in Human Stemness and Differentiation.

Fatemeh Mirzadeh Azad, Isabelle Laurence Polignano, Valentina Proserpio, Salvatore Oliviero.

  There is increasing evidence that long noncoding RNAs (lncRNAs) are among the main regulatory factors of stem cell maintenance and differentiation. They act through various mechanisms and interactions with proteins, DNA, and RNA. This heterogeneity in function increases the capabilities of the lncRNome toolkit but also makes it difficult to predict the function of novel lncRNAs or even rely on biological information produced in animal models. As lncRNAs are species- and tissue-specific, the recent technical advances in self-renewal and differentiation of human embryonic stem cells (ESCs) make these cells the ideal system to identify key regulatory lncRNAs and study their molecular functions. Here we provide an overview of the functional versatility of lncRNA mechanistic heterogeneity in regulating pluripotency maintenance and human differentiation.

Keywords:  endoderm; human pluripotent stem cells; long noncoding RNAs; mesoderm; neuroectoderm

DOI:  https://doi.org/10.1016/j.tcb.2021.02.002
Nat Commun. 2021 03 01. 12(1): 1366

Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics.

Lars Velten, Benjamin A Story, Pablo Hernández-Malmierca, Simon Raffel, Daniel R Leonce, Jennifer Milbank, Malte Paulsen, Aykut Demir, Chelsea Szu-Tu, Robert Frömel, Christoph Lutz, Daniel Nowak, Johann-Christoph Jann, Caroline Pabst, Tobias Boch, Wolf-Karsten Hofmann, Carsten Müller-Tidow, Andreas Trumpp, Simon Haas, Lars M Steinmetz.

Cancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

DOI: https://doi.org/10.1038/s41467-021-21650-1
Orphanet J Rare Dis. 2021 Feb 27. 16(1): 109

Clinical experience with the AKT1 inhibitor miransertib in two children with PIK3CA-related overgrowth syndrome.

Karina Forde, Nicoletta Resta, Carlotta Ranieri, David Rea, Olga Kubassova, Mark Hinton, Katrina A Andrews, Robert Semple, Alan D Irvine, Veronika Dvorakova.

   BACKGROUND: PIK3CA-related overgrowth spectrum (PROS) refers to a group of rare disorders, caused by somatic activating mutations in PIK3CA, resulting in abnormal PI3K-AKT-mTOR pathway signalling. Significant associated morbidity is frequently observed, and approved treatments are lacking. Miransertib (ARQ 092) is a novel, orally available, selective pan-AKT inhibitor with proven in vitro efficacy. Following recent results of the use of AKT inhibitors in Proteus syndrome (PS) and AKT-mutant cancers, we investigated its therapeutic use in two patients with severe PROS who had exhausted conventional treatment methods.
RESULTS: Two patients, one with CLOVES variant (P1) and one with facial infiltrating lipomatosis and hemimegalencephaly (P2), were commenced on miransertib treatment on a compassionate use basis. In patient one, intra-abdominal and paraspinal overgrowth had resulted in respiratory compromise, obstructive uropathy, dysfunctional seating and lying postures, and chronic pain. In patient two, hemifacial overgrowth and hemimegalencephaly had caused difficulties with articulation and oral function, and refractory epilepsy. Miransertib treatment was continued for a median duration of 22 months (range 22-28). In patient one, alleviation of respiratory compromise was observed and functionally, seating and lying postures improved. Serial volumetric MRI analysis revealed 15% reduction in calculated volumes of fatty overgrowth between treatment commencement and end. In patient two, reduction in seizure burden and improved parent-reported quality of life measures were reported. Treatment was discontinued in both patients due to lack of sustained response, and poor compliance in year two of treatment (P2). No significant toxicities were reported.
CONCLUSION: We report the first paediatric case series of the use of miransertib in two children with PROS. Objective clinical response was observed in patient one, and improvement in key qualitative outcomes was reported in patient two. Treatment was well tolerated with no significant toxicities reported. This case series highlights the potential therapeutic utility of miransertib in selected paediatric patients with severe PROS, and further demonstrates the potential for re-purposing targeted therapies for the treatment of rare diseases. An open label, Phase 1/2 study of miransertib in children with PROS and PS is underway to more accurately assess the efficacy of miransertib in the treatment of PROS disorder (NCT03094832).

Keywords:  AKT inhibitor; Miransertib; Next generation sequencing (NGS); PIK3CA-related overgrowth spectrum (PROS); Targeted treatment

DOI:  https://doi.org/10.1186/s13023-021-01745-0
Genes (Basel). 2021 Feb 17. pii: 285. [Epub ahead of print]12(2):

Molecular Biomarkers for Contemporary Therapies in Hormone Receptor-Positive Breast Cancer.

Allegra Freelander, Lauren J Brown, Andrew Parker, Davendra Segara, Neil Portman, Brandon Lau, Elgene Lim.

  Systemic treatment of hormone receptor-positive (HR+) breast cancer is undergoing a renaissance, with a number of targeted therapies including CDK4/6, mTOR, and PI3K inhibitors now approved for use in combination with endocrine therapies. The increased use of targeted therapies has changed the natural history of HR+ breast cancers, with the emergence of new escape mechanisms leading to the inevitable progression of disease in patients with advanced cancers. The identification of new predictive and pharmacodynamic biomarkers to current standard-of-care therapies and discovery of new therapies is an evolving and urgent clinical challenge in this setting. While traditional, routinely measured biomarkers such as estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth factor receptor 2 (HER2) still represent the best prognostic and predictive biomarkers for HR+ breast cancer, a significant proportion of patients either do not respond to endocrine therapy or develop endocrine resistant disease. Genomic tests have emerged as a useful adjunct prognostication tool and guide the addition of chemotherapy to endocrine therapy. In the treatment-resistant setting, mutational profiling has been used to identify ESR1, PIK3CA, and AKT mutations as predictive molecular biomarkers to newer therapies. Additionally, pharmacodynamic biomarkers are being increasingly used and considered in the metastatic setting. In this review, we summarise the current state-of-the-art therapies; prognostic, predictive, and pharmacodynamic molecular biomarkers; and how these are impacted by emerging therapies for HR+ breast cancer.

Keywords:  biomarkers; breast cancer; estrogen receptor; pharmacodynamic; predictive; prognostic

DOI:  https://doi.org/10.3390/genes12020285
Cell Metab. 2021 Mar 02. pii: S1550-4131(21)00067-X. [Epub ahead of print]33(3): 462-463

How does obesity promote breast cancer tumor growth?

Marc L Reitman.

Obesity is a risk factor for many cancers. Maguire et al. (2021) found increased creatine synthesis by the adipocytes adjacent to breast cancers in obese mice. The creatine is transported into the cancer cells, producing larger tumors, possibly due to greater energy availability.

DOI: https://doi.org/10.1016/j.cmet.2021.02.011
Nat Rev Clin Oncol. 2021 Mar 02.

Cancer therapies based on targeted protein degradation - lessons learned with lenalidomide.

Max Jan, Adam S Sperling, Benjamin L Ebert.

For decades, anticancer targeted therapies have been designed to inhibit kinases or other enzyme classes and have profoundly benefited many patients. However, novel approaches are required to target transcription factors, scaffolding proteins and other proteins central to cancer biology that typically lack catalytic activity and have remained mostly recalcitrant to drug development. The selective degradation of target proteins is an attractive approach to expand the druggable proteome, and the selective oestrogen receptor degrader fulvestrant served as an early example of this concept. Following a long and tragic history in the clinic, the immunomodulatory imide drug (IMiD) thalidomide was discovered to exert its therapeutic activity via a novel and unexpected mechanism of action: targeting proteins to an E3 ubiquitin ligase for subsequent proteasomal degradation. This discovery has paralleled and directly catalysed myriad breakthroughs in drug development, leading to the rapid maturation of generalizable chemical platforms for the targeted degradation of previously undruggable proteins. Decades of clinical experience have established front-line roles for thalidomide analogues, including lenalidomide and pomalidomide, in the treatment of haematological malignancies. With a new generation of 'degrader' drugs currently in development, this experience provides crucial insights into class-wide features of degraders, including a unique pharmacology, mechanisms of resistance and emerging therapeutic opportunities. Herein, we review these past experiences and discuss their application in the clinical development of novel degrader therapies.

DOI: https://doi.org/10.1038/s41571-021-00479-z
Trends Cancer. 2021 Feb 26. pii: S2405-8033(21)00044-3. [Epub ahead of print]

Quantifying Cancer: More Than Just a Numbers Game.

Ravi Salgia, Prakash Kulkarni, Herbert Levine, Danielle T Loughlin.

DOI: https://doi.org/10.1016/j.trecan.2021.02.002
Front Cell Dev Biol. 2021 ;9 626404

Akt Is S-Palmitoylated: A New Layer of Regulation for Akt.

Matías Blaustein, Estefanía Piegari, Camila Martínez Calejman, Antonella Vila, Analía Amante, María Victoria Manese, Ari Zeida, Laurence Abrami, Mariela Veggetti, David A Guertin, F Gisou van der Goot, María Martha Corvi, Alejandro Colman-Lerner.

  The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death.

Keywords:  Akt; Golgi; S-palmitoylation; autophagy; cell differentiation; cell signaling; lysosomes; subcellular localization

DOI:  https://doi.org/10.3389/fcell.2021.626404
Cell Stem Cell. 2021 Mar 04. pii: S1934-5909(21)00066-7. [Epub ahead of print]28(3): 409-423

Challenges in Studying Stem Cell Metabolism.

Cesar A Perez-Ramirez, Heather R Christofk.

The expanding field of stem cell metabolism has been supported by technical advances in metabolite profiling and novel functional analyses. While use of these methodologies has been fruitful, many challenges are posed by the intricacies of culturing stem cells in vitro, along with the distinctive scarcity of adult tissue stem cells and the complexities of their niches in vivo. This review provides an examination of the methodologies used to characterize stem cell metabolism, highlighting their utility while placing a sharper focus on their limitations and hurdles the field needs to overcome for the optimal study of stem cell metabolic networks.

DOI: https://doi.org/10.1016/j.stem.2021.02.016
Mol Metab. 2021 Feb 26. pii: S2212-8778(21)00037-5. [Epub ahead of print] 101197

Adipocyte Integrin-Linked Kinase Plays a Key Role in the Development of Diet-Induced Adipose Insulin Resistance in Male Mice.

Aimée R Bugler-Lamb, Annie Hasib, Xiong Weng, Chandani K Hennayake, Chenshi Lin, Rory J McCrimmon, Roland H Stimson, Michael L J Ashford, David H Wasserman, Li Kang.

   OBJECTIVE: Increased deposition of the extracellular matrix (ECM) in adipose tissue (AT) during obesity contributes to insulin resistance. The integrin receptors transmit changes in the extracellular environment causing corresponding intracellular adaptations. Integrin-linked kinase (ILK), an adaptor protein, is a central hub for intracellular signalling of integrins. This study determined the role of ILK in adipose function and insulin resistance.
METHODS: The pathogenic role of ILK in obesity and insulin resistance was studied in human adipose tissue as well as in adipocyte-specific ILK deficient mice (ILKlox/loxAdCre). ILKlox/loxAdCre mice together with wildtype littermates (ILKlox/lox) were fed with chow diet or 60% high fat (HF) diet for 16 weeks. In vivo insulin sensitivity was determined by the hyperinsulinemic-euglycemic clamp.
RESULTS: AT ILK expression was increased by HF diet feeding in mice and in visceral fat of morbidly obese humans. HF-fed ILKlox/loxAdCre mice displayed reduced fat mass and improved glucose tolerance relative to HF-fed ILKlox/lox mice. During a hyperinsulinemic-euglycemic clamp, HF-fed ILKlox/loxAdCre mice exhibited partially improved insulin resistance in AT. Lipolysis was suppressed to greater extent by insulin and glucose uptake in brown AT (BAT) was increased. Increased inhibition of lipolysis may be attributed to increased vascularization in white AT, while increased glucose uptake in BAT was associated with increased Akt phosphorylation and P38/JNK de-phosphorylation. Notably AT insulin sensitivity in lean mice was not affected by ILK deletion. Moreover, reduced fat mass in HF-fed ILKlox/loxAdCre mice may be attributed to decreased free fatty acid uptake into adipocytes via the down-regulation of CD36 gene expression. In consistent with results in mice, knockdown and knockout of ILK in 3T3-L1 cells decreased lipid accumulation and CD36 gene expression during adipogenesis.
CONCLUSIONS: These data show that adipocyte ILK is important in regulating HF diet-mediated insulin resistance in AT in a manner consistent with AT function.

Keywords:  Adipose tissue; Extracellular matrix; Insulin clamp; Insulin resistance; Integrin-linked kinase

DOI:  https://doi.org/10.1016/j.molmet.2021.101197
J Cell Biol. 2021 May 03. pii: e202006174. [Epub ahead of print]220(5):

Reduced insulin/IGF1 signaling prevents immune aging via ZIP-10/bZIP-mediated feedforward loop.

Yujin Lee, Yoonji Jung, Dae-Eun Jeong, Wooseon Hwang, Seokjin Ham, Hae-Eun H Park, Sujeong Kwon, Jasmine M Ashraf, Coleen T Murphy, Seung-Jae V Lee.

A hallmark of aging is immunosenescence, a decline in immune functions, which appeared to be inevitable in living organisms, including Caenorhabditis elegans. Here, we show that genetic inhibition of the DAF-2/insulin/IGF-1 receptor drastically enhances immunocompetence in old age in C. elegans. We demonstrate that longevity-promoting DAF-16/FOXO and heat-shock transcription factor 1 (HSF-1) increase immunocompetence in old daf-2(-) animals. In contrast, p38 mitogen-activated protein kinase 1 (PMK-1), a key determinant of immunity, is only partially required for this rejuvenated immunity. The up-regulation of DAF-16/FOXO and HSF-1 decreases the expression of the zip-10/bZIP transcription factor, which in turn down-regulates INS-7, an agonistic insulin-like peptide, resulting in further reduction of insulin/IGF-1 signaling (IIS). Thus, reduced IIS prevents immune aging via the up-regulation of anti-aging transcription factors that modulate an endocrine insulin-like peptide through a feedforward mechanism. Because many functions of IIS are conserved across phyla, our study may lead to the development of strategies against immune aging in humans.

DOI: https://doi.org/10.1083/jcb.202006174
Nat Rev Cancer. 2021 Mar 01.

MYC inhibition, courtesy of E. coli.

Karen O'Leary.

DOI: https://doi.org/10.1038/s41568-021-00345-1
Genes Dev. 2021 Mar 04.

Fine-tuning p53 activity by modulating the interaction between eukaryotic translation initiation factor eIF4E and RNA-binding protein RBM38.

Wenqiang Sun, Kyra Laubach, Christopher Lucchessi, Yanhong Zhang, Mingyi Chen, Jin Zhang, Xinbin Chen.

  p53 is critical for tumor suppression but also elicits detrimental effects when aberrantly overexpressed. Thus, multiple regulators, including RNA-binding protein RBM38, are found to tightly control p53 expression. Interestingly, RBM38 is unique in that it can either suppress or enhance p53 mRNA translation via altered interaction with eIF4E potentially mediated by serine-195 (S195) in RBM38. Thus, multiple RBM38/eIF4E knock-in (KI) cell lines were generated to investigate the significance of eIF4E-RBM38 interaction in controlling p53 activity. We showed that KI of RBM38-S195D or -Y192C enhances, whereas KI of RBM38-S195K/R/L weakens, the binding of eIF4E to p53 mRNA and subsequently p53 expression. We also showed that KI of eIF4E-D202K weakens the interaction of eIF4E with RBM38 and thereby enhances p53 expression, suggesting that D202 in eIF4E interacts with S195 in RBM38. Moreover, we generated an Rbm38 S193D KI mouse model in which human-equivalent serine-193 is substituted with aspartic acid. We showed that S193D KI enhances p53-dependent cellular senescence and that S193D KI mice have a shortened life span and are prone to spontaneous tumors, chronic inflammation, and liver steatosis. Together, we provide in vivo evidence that the RBM38-eIF4E loop can be explored to fine-tune p53 expression for therapeutic development.

Keywords:  RBM38; eIF4E; knock-in; p53; tumor suppression

DOI:  https://doi.org/10.1101/gad.346148.120
Cell Metab. 2021 Feb 23. pii: S1550-4131(21)00061-9. [Epub ahead of print]

CRISPR screens in physiologic medium reveal conditionally essential genes in human cells.

Nicholas J Rossiter, Kimberly S Huggler, Charles H Adelmann, Heather R Keys, Ross W Soens, David M Sabatini, Jason R Cantor.

  Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly reflect metabolite availability in human blood. Here, we performed CRISPR-based screens in traditional versus human plasma-like medium (HPLM). Sets of conditionally essential genes in human cancer cell lines span several cellular processes and vary with both natural cell-intrinsic diversity and the combination of basal and serum components that comprise typical media. Notably, we traced the causes for each of three conditional CRISPR phenotypes to the availability of metabolites uniquely defined in HPLM versus conventional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.

Keywords:  CRISPR; HPLM; conditional gene essentiality; gene-nutrient interaction; genetic screen; physiologic medium

DOI:  https://doi.org/10.1016/j.cmet.2021.02.005