bims-pideca 2023-02-26 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2023–02–26
forty-two papers selected by
Ralitsa Radostinova Madsen, University College London

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance.
Optogenetic decoding of Akt2-regulated metabolic signaling pathways in skeletal muscle cells using transomics analysis.
Nuclear PTEN's Functions in Suppressing Tumorigenesis: Implications for Rare Cancers.
Activated PI3K Delta Syndrome-1 mutations cause neutrophilia in zebrafish larvae.
AKT blocks SIK1-mediated repression of STAT3 to promote breast tumorigenesis.
PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells.
A versatile, high-efficiency platform for CRISPR-based gene activation.
The adaptor protein VEPH1 interacts with the kinase domain of ERBB2 and impacts EGF signaling in ovarian cancer cells.
Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition.
Reduced insulin signaling in neurons induces sex-specific health benefits.
Recurrent PIK3CA H1047R-Mutated Congenital Infiltrative Facial Lipomatosis: A Case Report and Review of Literature.
An interactive web application for processing, correcting, and visualizing genome-wide pooled CRISPR-Cas9 screens.
Insulin-mTOR hyperfunction drives C. elegans aging opposed by the megaprotein LPD-3.
The lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope binding.
Endothelial phosphoinositide 3-kinase-β inactivation confers protection from immune-mediated vascular injury.
PTEN phosphatase inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6.
Co-occurrence of Proteus syndrome and ventricular tachycardia cardiac arrest in a teenager.
Intra-ischemic hypothermia cardioprotection involves modulation of PTEN/Akt/ERK signaling and fatty acid oxidation.
Massively Parallel Profiling of Accessible Chromatin and Proteins with ASAP-Seq.
HaloTag-based reporters for fluorescence imaging and biosensing.
Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research.
Bilateral Wilms Tumor in CLOVES Syndrome.
Phenotypic pliancy and the breakdown of epigenetic polycomb mechanisms.
A pillar/perfusion plate enhances cell growth, reproducibility, throughput, and user friendliness in dynamic 3D cell culture.
Comprehensive chromatin proteomics resolves functional phases of pluripotency and identifies changes in regulatory components.
Pan-cancer clinical impact of latent drivers from double mutations.
Pixelated Microfluidics for Drug Screening on Tumour Spheroids and Ex Vivo Microdissected Tumour Explants.
Oscillatory ERK Signaling and Morphology Determine Heterogeneity of Breast Cancer Cell Chemotaxis via MEK-ERK and p38-MAPK Signaling Pathways.
Daily briefing: How artificial intelligence might change mathematics.
An angled-shape tip-based strategy for highly sensitive proteomic profiling of a low number of cells.
Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.
Identifying key multifunctional components shared by critical cancer and normal liver pathways via SparseGMM.
A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells.
Kinome inhibition states and multiomics data enable prediction of cell viability in diverse cancer types.
A conversation about building a support network in science.
Leading edge competition promotes context-dependent responses to receptor inputs to resolve directional dilemmas in neutrophil migration.
In silico discovery of small molecules for efficient stem cell differentiation into definitive endoderm.
Network-based elucidation of colon cancer drug resistance by phosphoproteomic time-series analysis.
Nuclear export inhibition jumbles epithelial-mesenchymal states and gives rise to migratory disorder in healthy epithelia.
Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.
Endogenous DAF-16 spatiotemporal activity quantitatively predicts lifespan extension induced by dietary restriction.
Large-scale organoid study suggests effects of trisomy 21 on early fetal neurodevelopment are more subtle than variability between isogenic lines and experiments.

Nat Commun. 2023 Feb 18. 14(1): 923

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance.

Daniel J Fazakerley, Julian van Gerwen, Kristen C Cooke, Xiaowen Duan, Elise J Needham, Alexis Díaz-Vegas, Søren Madsen, Dougall M Norris, Amber S Shun-Shion, James R Krycer, James G Burchfield, Pengyi Yang, Mark R Wade, Joseph T Brozinick, David E James, Sean J Humphrey.

The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employ phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults causing insulin resistance, we observe a marked rewiring of the insulin signaling network. This includes both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying dysregulated phosphosites common to multiple insults reveals subnetworks containing non-canonical regulators of insulin action, such as MARK2/3, and causal drivers of insulin resistance. The presence of several bona fide GSK3 substrates among these phosphosites led us to establish a pipeline for identifying context-specific kinase substrates, revealing widespread dysregulation of GSK3 signaling. Pharmacological inhibition of GSK3 partially reverses insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that includes dysregulated MARK2/3 and GSK3 activity.

DOI: https://doi.org/10.1038/s41467-023-36549-2
Sci Signal. 2023 Feb 21. 16(773): eabn0782

Optogenetic decoding of Akt2-regulated metabolic signaling pathways in skeletal muscle cells using transomics analysis.

Genki Kawamura, Toshiya Kokaji, Kentaro Kawata, Yuka Sekine, Yutaka Suzuki, Tomoyoshi Soga, Yoshibumi Ueda, Mizuki Endo, Shinya Kuroda, Takeaki Ozawa.

Insulin regulates various cellular metabolic processes by activating specific isoforms of the Akt family of kinases. Here, we elucidated metabolic pathways that are regulated in an Akt2-dependent manner. We constructed a transomics network by quantifying phosphorylated Akt substrates, metabolites, and transcripts in C2C12 skeletal muscle cells with acute, optogenetically induced activation of Akt2. We found that Akt2-specific activation predominantly affected Akt substrate phosphorylation and metabolite regulation rather than transcript regulation. The transomics network revealed that Akt2 regulated the lower glycolysis pathway and nucleotide metabolism and cooperated with Akt2-independent signaling to promote the rate-limiting steps in these processes, such as the first step of glycolysis, glucose uptake, and the activation of the pyrimidine metabolic enzyme CAD. Together, our findings reveal the mechanism of Akt2-dependent metabolic pathway regulation, paving the way for Akt2-targeting therapeutics in diabetes and metabolic disorders.

DOI: https://doi.org/10.1126/scisignal.abn0782
Biomolecules. 2023 Jan 30. pii: 259. [Epub ahead of print]13(2):

Nuclear PTEN's Functions in Suppressing Tumorigenesis: Implications for Rare Cancers.

Casey G Langdon.

  Phosphatase and tensin homolog (PTEN) encodes a tumor-suppressive phosphatase with both lipid and protein phosphatase activity. The tumor-suppressive functions of PTEN are lost through a variety of mechanisms across a wide spectrum of human malignancies, including several rare cancers that affect pediatric and adult populations. Originally discovered and characterized as a negative regulator of the cytoplasmic, pro-oncogenic phosphoinositide-3-kinase (PI3K) pathway, PTEN is also localized to the nucleus where it can exert tumor-suppressive functions in a PI3K pathway-independent manner. Cancers can usurp the tumor-suppressive functions of PTEN to promote oncogenesis by disrupting homeostatic subcellular PTEN localization. The objective of this review is to describe the changes seen in PTEN subcellular localization during tumorigenesis, how PTEN enters the nucleus, and the spectrum of impacts and consequences arising from disrupted PTEN nuclear localization on tumor promotion. This review will highlight the immediate need in understanding not only the cytoplasmic but also the nuclear functions of PTEN to gain more complete insights into how important PTEN is in preventing human cancers.

Keywords:  DNA damage; PTEN; PTEN hamartoma tumor syndrome; genetically engineered mouse models; nuclear PTEN; nuclear import; oncogenic transcriptional regulation; post-translational modifications; subcellular localization

DOI:  https://doi.org/10.3390/biom13020259
Dis Model Mech. 2023 Feb 20. pii: dmm.049841. [Epub ahead of print]

Activated PI3K Delta Syndrome-1 mutations cause neutrophilia in zebrafish larvae.

Stone Elworthy, Holly A Rutherford, Tomasz K Prajsnar, Noémie M Hamilton, Katja Vogt, Stephen A Renshaw, Alison M Condliffe.

  People with Activated PI3 Kinase Delta Syndrome 1 (APDS1) suffer from immune deficiency and severe bronchiectasis. APDS1 is caused by dominant activating mutations of the PIK3CD gene that encodes the PI3 kinase delta (PI3Kδ) catalytic subunit. Despite the importance of innate immunity defects in bronchiectasis, there has been limited investigation of neutrophils or macrophages in APDS1 patients or mouse models. Zebrafish embryos provide an ideal system to study neutrophils and macrophages. Previous studies of zebrafish with strongly hyperactivated PI3 kinase activity due to Pten deficiency, revealed excessive production of immature neutrophils that fail to mature. We used CRISPR-Cas9 and CRISPR-Cpf1, with oligo-nucleotide directed homologous repair, to engineer zebrafish equivalents of the two most prevalent human APDS1 disease mutations. These zebrafish pik3cd alleles dominantly cause excessive neutrophilic inflammation in a tail-fin injury model. They also exhibit total body neutrophilia in the absence of any inflammatory stimulus but have normal numbers of macrophages. Exposure to the PI3Kδ inhibitor CAL-101 reverses the total body neutrophilia. There is no apparent defect in neutrophil maturation or migration and tail-fin regeneration is unimpaired.

Keywords:  APDS; CRISPR; Cas12a; Cpf1; Homology-directed gene editing; Neutrophil; Neutrophilia; PI3K-delta; Zebrafish

DOI:  https://doi.org/10.1242/dmm.049841
Cancer Res. 2023 Feb 20. pii: CAN-22-3407. [Epub ahead of print]

AKT blocks SIK1-mediated repression of STAT3 to promote breast tumorigenesis.

Zhicheng Sun, Qiwei Jiang, Bing Gao, Xiaomei Zhang, Lang Bu, Lei Wang, Ying Lin, Wei Xie, Jie Li, Jianping Guo.

The PI3K-AKT signaling pathway is frequently dysregulated in cancer, and it is hyperactivated in approximately 50% of breast cancers. While inhibitors directly targeting the PI3K-AKT axis have been developed, clinical efficacy has been limited to only a subset of patients. Identification of mechanisms underlying AKT-driven tumorigenesis could lead to alternative approaches to block pathway signaling and suppress breast tumor growth. Mass spectrometry-based analyses demonstrated that salt-inducible kinase 1 (SIK1) binds AKT and undergoes AKT-mediated phosphorylation, which compromises SIK1 tumor-suppressive functions. As a result, AKT relieved the binding and repression of STAT3 by SIK1 in a phosphorylation-dependent manner, resulting in breast cell tumorigenesis. Following AKT-mediated phosphorylation, SIK1 interacted with 14-3-3 and was translocated to the cytoplasm where the isomerase Pin1 facilitated SIK1 interaction with the E3 ligase ITCH to promote SIK1 ubiquitination and subsequent degradation. These findings indicate that SIK1 is a substrate of AKT that links AKT oncogenic function to STAT3 activation, highlighting targeting of the JAK2-STAT3 axis as a strategy to treat AKT-driven breast cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-3407
Sci Adv. 2023 Feb 22. 9(8): eade8222

PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells.

Kristina Ames, Imit Kaur, Yang Shi, Meng M Tong, Taneisha Sinclair, Shayda Hemmati, Shira G Glushakow-Smith, Ellen Tein, Lindsay Gurska, Ulrich Steidl, Robert Dubin, Jidong Shan, Cristina Montagna, Kith Pradhan, Amit Verma, Kira Gritsman.

Myelodysplastic syndrome (MDS) is a clonal malignancy arising in hematopoietic stem cells (HSCs). The mechanisms of MDS initiation in HSCs are still poorly understood. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is frequently activated in acute myeloid leukemia, but in MDS, PI3K/AKT is often down-regulated. To determine whether PI3K down-regulation can perturb HSC function, we generated a triple knockout (TKO) mouse model with Pik3ca, Pik3cb, and Pik3cd deletion in hematopoietic cells. Unexpectedly, PI3K deficiency caused cytopenias, decreased survival, and multilineage dysplasia with chromosomal abnormalities, consistent with MDS initiation. TKO HSCs exhibit impaired autophagy, and pharmacologic autophagy induction improved HSC differentiation. Using intracellular LC3 and P62 flow cytometry and transmission electron microscopy, we also observed abnormal autophagic degradation in patient MDS HSCs. Therefore, we have uncovered an important protective role for PI3K in maintaining autophagic flux in HSCs to preserve the balance between self-renewal and differentiation and to prevent MDS initiation.

DOI: https://doi.org/10.1126/sciadv.ade8222
Nat Commun. 2023 Feb 17. 14(1): 902

A versatile, high-efficiency platform for CRISPR-based gene activation.

Amy J Heidersbach, Kristel M Dorighi, Javier A Gomez, Ashley M Jacobi, Benjamin Haley.

CRISPR-mediated transcriptional activation (CRISPRa) is a powerful technology for inducing gene expression from endogenous loci with exciting applications in high throughput gain-of-function genomic screens and the engineering of cell-based models. However, current strategies for generating potent, stable, CRISPRa-competent cell lines present limitations for the broad utility of this approach. Here, we provide a high-efficiency, self-selecting CRISPRa enrichment strategy, which combined with piggyBac transposon technology enables rapid production of CRISPRa-ready cell populations compatible with a variety of downstream assays. We complement this with an optimized guide RNA scaffold that significantly enhances CRISPRa functionality. Finally, we describe a synthetic guide RNA tool set that enables transient, population-wide gene activation when used with the self-selecting CRISPRa system. Taken together, this versatile platform greatly enhances the potential for CRISPRa across a wide variety of cellular contexts.

DOI: https://doi.org/10.1038/s41467-023-36452-w
Cell Signal. 2023 Feb 22. pii: S0898-6568(23)00048-7. [Epub ahead of print] 110634

The adaptor protein VEPH1 interacts with the kinase domain of ERBB2 and impacts EGF signaling in ovarian cancer cells.

Alexandra Kollara, Brian D Burt, Maurice J Ringuette, Theodore J Brown.

  Upregulation of ERBB2 and activating mutations in downstream KRAS/BRAF and PIK3CA are found in several ovarian cancer histotypes. ERBB2 enhances signaling by the ERBB family of EGF receptors, and contains docking positions for proteins that transduce signaling through multiple pathways. We identified the adaptor protein ventricular zone-expressed pleckstrin homology domain-containing protein 1 (VEPH1) as a potential interacting partner of ERBB2 in a screen of proteins co-immunoprecipitated with VEPH1. In this study, we confirm a VEPH1 - ERBB2 interaction by co-immunoprecipitation and biotin proximity labelling and show that VEPH1 interacts with the juxtamembrane-kinase domain of ERBB2. In SKOV3 ovarian cancer cells, which bear a PIK3CA mutation and ERBB2 overexpression, ectopic VEPH1 expression enhanced EGF activation of ERK1/2, and mTORC2 activation of AKT. In contrast, in ES2 ovarian cancer cells, which bear a BRAFV600E mutation with VEPH1 amplification but low ERBB2 expression, loss of VEPH1 expression enabled further activation of ERK1/2 by EGF and enhanced EGF activation of AKT. VEPH1 expression in SKOV3 cells enhanced EGF-induced cell migration consistent with increased Snail2 and decreased E-cadherin levels. In comparison, loss of VEPH1 expression in ES2 cells led to decreased cell motility independent of EGF treatment despite higher levels of N-cadherin and Snail2. Importantly, we found that loss of VEPH1 expression rendered ES2 cells less sensitive to BRAF and MEK inhibition. This study extends the range of adaptor function of VEPH1 to ERBB2, and indicates VEPH1 has differential effects on EGF signaling in ovarian cancer cells that may be influenced by driver gene mutations.

Keywords:  AKT; EGF; ERBB2; ERK1/2; Ovarian cancer; VEPH1

DOI:  https://doi.org/10.1016/j.cellsig.2023.110634
J Exp Med. 2023 May 01. pii: e20221563. [Epub ahead of print]220(5):

Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition.

Wei Wei, Mitchell J Geer, Xinyi Guo, Igor Dolgalev, Neville E Sanjana, Benjamin G Neel.

SHP2 (PTPN11) acts upstream of SOS1/2 to enable RAS activation. Allosteric SHP2 inhibitors (SHP2i) in the clinic prevent SHP2 activation, block proliferation of RTK- or cycling RAS mutant-driven cancers, and overcome "adaptive resistance." To identify SHP2i resistance mechanisms, we performed genome-wide CRISPR/Cas9 knockout screens on two SHP2i-sensitive cell lines, recovering genes expected to cause resistance (NF1, PTEN, CDKN1B, LZTR1, and RASA2) and novel targets (INPPL1, MAP4K5, epigenetic modifiers). We screened 14 additional lines with a focused CRISPR library targeting common "hits" from the genome-wide screens. LZTR1 deletion conferred resistance in 12/14 lines, followed by MAP4K5 (8/14), SPRED2/STK40 (6/14), and INPPL1 (5/14). INPPL1, MAP4K5, or LZTR1 deletion reactivated ERK signaling. INPPL1-mediated sensitization to SHP2i required its NPXY motif but not lipid phosphatase activity. MAP4K5 acted upstream of MEK through a kinase-dependent target(s); LZTR1 had cell-dependent effects on RIT and RAS stability. INPPL1, MAP4K5, or LZTR1 deletion also conferred SHP2i resistance in vivo. Defining the SHP2i resistance landscape could suggest effective combination approaches.

DOI: https://doi.org/10.1084/jem.20221563
Sci Adv. 2023 Feb 22. 9(8): eade8137

Reduced insulin signaling in neurons induces sex-specific health benefits.

Maarouf Baghdadi, Tobias Nespital, Andrea Mesaros, Sandra Buschbaum, Dominic J Withers, Sebastian Grönke, Linda Partridge.

Reduced activity of insulin/insulin-like growth factor signaling (IIS) extends health and life span in mammals. Loss of the insulin receptor substrate 1 (Irs1) gene increases survival in mice and causes tissue-specific changes in gene expression. However, the tissues underlying IIS-mediated longevity are currently unknown. Here, we measured survival and health span in mice lacking IRS1 specifically in liver, muscle, fat, and brain. Tissue-specific loss of IRS1 did not increase survival, suggesting that lack of IRS1 in more than one tissue is required for life-span extension. Loss of IRS1 in liver, muscle, and fat did not improve health. In contrast, loss of neuronal IRS1 increased energy expenditure, locomotion, and insulin sensitivity, specifically in old males. Neuronal loss of IRS1 also caused male-specific mitochondrial dysfunction, activation of Atf4, and metabolic adaptations consistent with an activated integrated stress response at old age. Thus, we identified a male-specific brain signature of aging in response to reduced IIS associated with improved health at old age.

DOI: https://doi.org/10.1126/sciadv.ade8137
Curr Issues Mol Biol. 2023 Feb 17. 45(2): 1712-1719

Recurrent PIK3CA H1047R-Mutated Congenital Infiltrative Facial Lipomatosis: A Case Report and Review of Literature.

Kei Shing Oh, Hisham F Bahmad, Kalin Veselinov Stoyanov, Ibrahim H Amjad, Carole Brathwaite.

  Congenital infiltrating lipomatosis of the face (CILF) is a rare, congenital, nonhereditary facial overgrowth due to post-zygomatic activating mutations in PIK3CA gene. It is unilateral and involves hypertrophy of both the soft and hard tissue structures on the affected side of the face. This commonly results in early eruption of the teeth, hypertrophy of the facial bones, macroglossia, and proliferation of the parotid gland. Less than 80 cases of CILF have been reported in the literature so far. Treatment modalities include liposuction and surgical excision. However, since the hallmark of CILF is mutation in the PIK3CA gene, PI3K inhibitors may play a therapeutic role in CILF. We report a case of an 8-year-old boy with recurrent CILF of the scalp and nose, with PIK3CA H1047R mutation. We discuss the differential diagnoses, clinical outcomes, and management of this rare entity.

Keywords:  PIK3CA; face; lipomatosis; surgery

DOI:  https://doi.org/10.3390/cimb45020110
Cell Rep Methods. 2023 Jan 23. 3(1): 100373

An interactive web application for processing, correcting, and visualizing genome-wide pooled CRISPR-Cas9 screens.

Alessandro Vinceti, Riccardo Roberto De Lucia, Paolo Cremaschi, Umberto Perron, Emre Karakoc, Luca Mauri, Carlos Fernandez, Krzysztof Henryk Kluczynski, Daniel Stephen Anderson, Francesco Iorio.

  A limitation of pooled CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes arising from copy-number-amplified genomics regions. To solve this issue, we previously developed CRISPRcleanR: a computational method implemented as R/python package and in a dockerized version. CRISPRcleanR detects and corrects biased responses to CRISPR-Cas9 targeting in an unsupervised fashion, accurately reducing false-positive signals while maintaining sensitivity in identifying relevant genetic dependencies. Here, we present CRISPRcleanR WebApp , a web application enabling access to CRISPRcleanR through an intuitive interface. CRISPRcleanR WebApp removes the complexity of R/python language user interactions; provides user-friendly access to a complete analytical pipeline, not requiring any data pre-processing and generating gene-level summaries of essentiality with associated statistical scores; and offers a range of interactively explorable plots while supporting a more comprehensive range of CRISPR guide RNAs' libraries than the original package. CRISPRcleanR WebApp is available at https://crisprcleanr-webapp.fht.org/.

Keywords:  CRISPR-Cas9 screens; bias correction; cancer dependency; copy number; data exploration; data visualization; gene essentiality; post-genomic data; unsupervised analysis; web application

DOI:  https://doi.org/10.1016/j.crmeth.2022.100373
bioRxiv. 2023 Feb 15. pii: 2023.02.14.528431. [Epub ahead of print]

Insulin-mTOR hyperfunction drives C. elegans aging opposed by the megaprotein LPD-3.

Taruna Pandey, Bingying Wang, Jenny Zu, Huichao Deng, Kang Shen, Goncalo Dias do Vale, Jeffrey G McDonald, Dengke K Ma.

Decreased insulin-mTOR signaling enables exceptional longevity in the nematode C. elegans by activating geroprotective transcription factors, including DAF-16, SKN-1 and HSF-1. Few studies have examined whether and how increased insulin-mTOR may actively drive organismic aging. Here we show that an agonist insulin INS-7 is drastically over-produced and causes shortened lifespan in lpd-3 mutants, a C. elegans model of human Alkuraya-Kuƒçinskas syndrome. Lipidomic profiling reveals marked increase in the abundance of hexaceramide species in lpd-3 mutants, consistent with up-regulation of the genes encoding biosynthetic enzymes for hexaceramides, including HYL-1 ( H omolog of Y east L ongevity). Reducing HYL-1 activity decreases INS-7 levels and rescues the shortened lifespan of lpd-3 mutants through InsR/DAF-2 and mTOR/LET-363. We propose that increased insulin signaling exhibits late-life antagonistic pleiotropy and shortens lifespans through sphingolipid-hexaceramide and mTOR regulatory pathways.

DOI: https://doi.org/10.1101/2023.02.14.528431
Nucleus. 2023 Dec;14(1): 2180206

The lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope binding.

Melanie Wallace, Gregory R Fedorchak, Richa Agrawal, Rachel M Gilbert, Jineet Patel, Sangwoo Park, Matthew Paszek, Jan Lammerding.

  Lamins A/C are nuclear intermediate filament proteins that are involved in diverse cellular mechanical and biochemical functions. Here, we report that recognition of Lamins A/C by a commonly used antibody (JOL-2) that binds the Lamin A/C Ig-fold and other antibodies targeting similar epitopes is highly dependent on cell density, even though Lamin A/Clevels do not change. We propose that the effect is caused by partial unfolding or masking of the C'E and/or EF loops of the Ig-fold in response to cell spreading. Surprisingly, JOL-2 antibody labeling was insensitive to disruption of cytoskeletal filaments or the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Furthermore, neither nuclear stiffness nor nucleo-cytoskeletal force transmission changed with cell density. These findings are important for the interpretation of immunofluorescence data for Lamin A/C and also raise the intriguing prospect that the conformational changes may play a role in Lamin A/C mediated cellular function.

Keywords:  Ig-fold; Lamins; cell density; cell spreading; immunofluorescence; immunolabeling; lamin A; lamin A/C; lamin C; mechanobiology; nuclear envelope

DOI:  https://doi.org/10.1080/19491034.2023.2180206
Am J Transplant. 2023 Feb;pii: S1600-6135(22)27253-0. [Epub ahead of print]23(2): 202-213

Endothelial phosphoinositide 3-kinase-β inactivation confers protection from immune-mediated vascular injury.

Andrew G Masoud, Jiaxin Lin, Lin F Zhu, Kesheng Tao, Nathan W Ness, Zamaneh Kassiri, Ronald B Moore, Bart Vanhaesebroeck, Lori West, Colin C Anderson, Gavin Y Oudit, Allan G Murray.

  Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-β (PI3Kβ) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kβ inhibitor-treated, or endothelial-selective PI3Kβ knockout (ECβKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kβ-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECβKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECβKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kβ inhibition or RNA interference. Selective PI3Kβ inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kβ as a therapeutic target to reduce vascular inflammation and injury.

Keywords:  animal models: murine; basic (laboratory) research/science; cellular biology; heart transplantation/cardiology; immunosuppressive regimens–rescue; rejection: chronic; rejection: vascular; signaling/signaling pathways: PI-3 kinase/AKT pathway; translational research/science

DOI:  https://doi.org/10.1016/j.ajt.2022.11.014
iScience. 2023 Feb 17. 26(2): 106070

PTEN phosphatase inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6.

Yanlin Yu, Meng Dai, Liping Huang, Weiping Chen, Ellen Yu, Arnulfo Mendoza, Helen Michael, Chand Khanna, Marcus Bosenberg, Martin McMahon, Glenn Merlino.

  PTEN encodes a tumor suppressor with lipid and protein phosphatase activities whose dysfunction has been implicated in melanomagenesis; less is known about how its phosphatases regulate melanoma metastasis. We demonstrate that PTEN expression negatively correlates with metastatic progression in human melanoma samples and a PTEN-deficient mouse melanoma model. Wildtype PTEN expression inhibited melanoma cell invasiveness and metastasis in a dose-dependent manner, behaviors that specifically required PTEN protein phosphatase activity. PTEN phosphatase activity regulated metastasis through Entpd5. Entpd5 knockdown reduced metastasis and IGF1R levels while promoting ER stress. In contrast, Entpd5 overexpression promoted metastasis and enhanced IGF1R levels while reducing ER stress. Moreover, Entpd5 expression was regulated by the ER stress sensor ATF6. Altogether, our data indicate that PTEN phosphatase activity inhibits metastasis by negatively regulating the Entpd5/IGF1R pathway through ATF6, thereby identifying novel candidate therapeutic targets for the treatment of PTEN mutant melanoma.

Keywords:  Cancer; Cell biology; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2023.106070
Am J Med Genet A. 2023 Feb 20.

Co-occurrence of Proteus syndrome and ventricular tachycardia cardiac arrest in a teenager.

Richard Ferguson, Ingrid Scurr, Christopher A Ours, Jennifer J Johnston, Katharine Pike, Georgia Spentzou.

  Proteus syndrome is an extremely rare overgrowth condition caused by a somatic variant of the AKT1 gene. It can involve multiple organ systems though rarely is there symptomatic cardiac involvement. Fatty infiltration of the myocardium has been described but has not been reported to cause functional or conduction abnormalities. We present an individual with Proteus syndrome who suffered a sudden cardiac arrest.

Keywords:  Proteus syndrome; cardiac arrest; ventricular tachycardia

DOI:  https://doi.org/10.1002/ajmg.a.63151
Physiol Rep. 2023 Feb;11(4): e15611

Intra-ischemic hypothermia cardioprotection involves modulation of PTEN/Akt/ERK signaling and fatty acid oxidation.

Cody N Justice, Xiangdong Zhu, Jing Li, J Michael O'Donnell, Terry L Vanden Hoek.

  Therapeutic hypothermia (TH) provides cardioprotection from ischemia/reperfusion (I/R) injury. However, it remains unknown how TH regulates metabolic recovery. We tested the hypothesis that TH modulates PTEN, Akt, and ERK1/2, and improves metabolic recovery through mitigation of fatty acid oxidation and taurine release. Left ventricular function was monitored continuously in isolated rat hearts subjected to 20 min of global, no-flow ischemia. Moderate cooling (30°C) was applied at the start of ischemia and hearts were rewarmed after 10 min of reperfusion. The effect of TH on protein phosphorylation and expression at 0 and 30 min of reperfusion was investigated by western blot analysis. Post-ischemic cardiac metabolism was investigated by 13 C-NMR. TH enhanced recovery of cardiac function, reduced taurine release, and enhanced PTEN phosphorylation and expression. Phosphorylation of Akt and ERK1/2 was increased at the end of ischemia but decreased at the end of reperfusion. On NMR analysis, TH-treated hearts displayed decreased fatty acid oxidation. Direct cardioprotection by moderate intra-ischemic TH is associated with decreased fatty acid oxidation, reduced taurine release, enhanced PTEN phosphorylation and expression, and enhanced activation of both Akt and ERK1/2 prior to reperfusion.

Keywords:  PTEN; fatty acid oxidation; ischemia/reperfusion; therapeutic hypothermia

DOI:  https://doi.org/10.14814/phy2.15611
Methods Mol Biol. 2023 ;2611 249-267

Massively Parallel Profiling of Accessible Chromatin and Proteins with ASAP-Seq.

Eleni P Mimitou, Peter Smibert, Caleb A Lareau.

  While methods such as the Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) enable a comprehensive characterization of regulatory DNA, additional measurements are required to characterize the multifaceted nature of eukaryotic cells. Here, we delineate the ATAC with Select Antigen Profiling by sequencing (ASAP-seq) protocol, a scalable approach to quantifying proteins via oligo-tagged antibodies alongside accessible DNA in thousands of single cells. Critically, our method utilizes a custom bridge oligo that enables the utilization of a variety of oligo-conjugated antibodies, enabling the utilization and repurposing of other commercial products. The ASAP-seq method can be completed with straightforward experimental and computational modifications existing single-cell ATAC-seq workflows but yields distinct modalities underlying complex cellular states, including estimation of protein abundance on the cell surface as well as intracellular and intranuclear factors.

Keywords:  ATAC; Accessible chromatin; Gene regulation; Intracellular; Multimodal; Protein; Single-cell

DOI:  https://doi.org/10.1007/978-1-0716-2899-7_13
Chembiochem. 2023 Feb 23. e202300022

HaloTag-based reporters for fluorescence imaging and biosensing.

Alexander Cook, Franziska Walterspiel, Claire Deo.

  Visualizing the structure and dynamics of biomolecules is critical to understand biological function, and requires methods to fluorescently label targets of interest in their cellular context. Self-labelling proteins, which combine a genetically encoded tag with a small-molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations of fluorescent proteins. Among them, the HaloTag protein is the most broadly used, showing fast specific labelling with a small, easy to functionalize and cell-permeant ligand. Synthetic chemistry and protein engineering have provided a portfolio of powerful imaging tools exploiting HaloTag, along with general methods to optimize and adapt them to specific applications. Here, we provide an overview of fluorescent reporters based on the HaloTag protein for imaging and biosensing, highlighting engineering strategies and general applications.

Keywords:  Fluorescent probes; Fluorogenicity; HaloTag; Self-labelling tags; biosensors

DOI:  https://doi.org/10.1002/cbic.202300022
FEBS Open Bio. 2023 Feb 22.

Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research.

Stefano L Giandomenico, Erin M Schuman.

  Gaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former approach is applicable to a wider range of targets, is more precise and can address more nuanced functional aspects. Despite such apparent advantages, genetic manipulation (i.e. knock-down, knock-out, mutation and tagging) in mammalian systems can be challenging due to problems with delivery, low rates of homologous recombination and epigenetic silencing. The advent of CRISPR-Cas9 in combination with the development of robust differentiation protocols that can efficiently generate a variety of different cell types in vitro has accelerated our ability to probe gene function in a more physiological setting. Often the main obstacle in this path of enquiry is to achieve the desired genetic modification. In this short review, we will focus on gene perturbation in mammalian cells and how editing and differentiation of pluripotent stem cells can complement more traditional approaches. Additionally, we introduce novel targeted protein degradation approaches as an alternative to DNA/RNA-based manipulation. Our aim is to provide a broad overview of recent approaches and in vitro systems to study mammalian cell biology. Due to space limitations, we limit ourselves to provide the inexperienced reader with a conceptual framework on how to use these tools, and for more in-depth information we will provide specific references throughout.

Keywords:  CRISPR; TPD; delivery; differentiation; neuroscience

DOI:  https://doi.org/10.1002/2211-5463.13581
Urology. 2023 Feb 16. pii: S0090-4295(23)00090-0. [Epub ahead of print]

Bilateral Wilms Tumor in CLOVES Syndrome.

Joon Kyung Kim, Anna Hansen, Leslie Peard, Matthew Newsome, Amanda F Saltzman.

  Wilms tumor (WT) is the most common pediatric renal mas and occurs in up to ten percent of predisposition syndromes. One such syndrome is CLOVES syndrome, an extremely rare disorder within the umbrella of PIK3CA-Related Overgrowth Spectrum (PROS) disorders. This case presents the management of bilateral WT in a patient with CLOVES syndrome and highlights the many intricacies in caring for complex oncology patients. Particularly highlighted in this case is the delicate line in balancing the risks of treatment-related morbidity against the risks of recurrence in predisposed patients, while still abiding by established treatment guidelines.

Keywords:  AREN0534; Bilateral Renal Masses; CLOVES Syndrome; PROS Disorders; Wilms Tumor

DOI:  https://doi.org/10.1016/j.urology.2023.01.034
PLoS Comput Biol. 2023 Feb 21. 19(2): e1010889

Phenotypic pliancy and the breakdown of epigenetic polycomb mechanisms.

Maryl Lambros, Yehonatan Sella, Aviv Bergman.

Epigenetic regulatory mechanisms allow multicellular organisms to develop distinct specialized cell identities despite having the same total genome. Cell-fate choices are based on gene expression programs and environmental cues that cells experience during embryonic development, and are usually maintained throughout the life of the organism despite new environmental cues. The evolutionarily conserved Polycomb group (PcG) proteins form Polycomb Repressive Complexes that help orchestrate these developmental choices. Post-development, these complexes actively maintain the resulting cell fate, even in the face of environmental perturbations. Given the crucial role of these polycomb mechanisms in providing phenotypic fidelity (i.e. maintenance of cell fate), we hypothesize that their dysregulation after development will lead to decreased phenotypic fidelity allowing dysregulated cells to sustainably switch their phenotype in response to environmental changes. We call this abnormal phenotypic switching phenotypic pliancy. We introduce a general computational evolutionary model that allows us to test our systems-level phenotypic pliancy hypothesis in-silico and in a context-independent manner. We find that 1) phenotypic fidelity is an emergent systems-level property of PcG-like mechanism evolution, and 2) phenotypic pliancy is an emergent systems-level property resulting from this mechanism's dysregulation. Since there is evidence that metastatic cells behave in a phenotypically pliant manner, we hypothesize that progression to metastasis is driven by the emergence of phenotypic pliancy in cancer cells as a result of PcG mechanism dysregulation. We corroborate our hypothesis using single-cell RNA-sequencing data from metastatic cancers. We find that metastatic cancer cells are phenotypically pliant in the same manner as predicted by our model.

DOI: https://doi.org/10.1371/journal.pcbi.1010889
bioRxiv. 2023 Feb 19. pii: 2023.02.16.528892. [Epub ahead of print]

A pillar/perfusion plate enhances cell growth, reproducibility, throughput, and user friendliness in dynamic 3D cell culture.

Vinod Kumar Reddy Lekkala, Soo-Yeon Kang, Jiafeng Liu, Sunil Shrestha, Prabha Acharya, Pranav Joshi, Mona Zolfaghar, Minseong Lee, Paarth Jamdagneya, Sohan Pagnis, Arham Kundi, Samarth Kabbur, Ung Tae Kim, Yong Yang, Moo-Yeal Lee.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from the necrotic core due to limited nutrient and oxygen diffusion and waste removal and have limited in vivo -like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids have been loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow rates were maintained within perfusion wells, and the pillar plate could be separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in dynamic 3D cell culture.

DOI: https://doi.org/10.1101/2023.02.16.528892
Nucleic Acids Res. 2023 Feb 20. pii: gkad058. [Epub ahead of print]

Comprehensive chromatin proteomics resolves functional phases of pluripotency and identifies changes in regulatory components.

Enes Ugur, Alexandra de la Porte, Weihua Qin, Sebastian Bultmann, Alina Ivanova, Micha Drukker, Matthias Mann, Michael Wierer, Heinrich Leonhardt.

The establishment of cellular identity is driven by transcriptional and epigenetic regulators of the chromatin proteome - the chromatome. Comprehensive analyses of the chromatome composition and dynamics can therefore greatly improve our understanding of gene regulatory mechanisms. Here, we developed an accurate mass spectrometry (MS)-based proteomic method called Chromatin Aggregation Capture (ChAC) followed by Data-Independent Acquisition (DIA) and analyzed chromatome reorganizations during major phases of pluripotency. This enabled us to generate a comprehensive atlas of proteomes, chromatomes, and chromatin affinities for the ground, formative and primed pluripotency states, and to pinpoint the specific binding and rearrangement of regulatory components. These comprehensive datasets combined with extensive analyses identified phase-specific factors like QSER1 and JADE1/2/3 and provide a detailed foundation for an in-depth understanding of mechanisms that govern the phased progression of pluripotency. The technical advances reported here can be readily applied to other models in development and disease.

DOI: https://doi.org/10.1093/nar/gkad058
Commun Biol. 2023 Feb 20. 6(1): 202

Pan-cancer clinical impact of latent drivers from double mutations.

Bengi Ruken Yavuz, Chung-Jung Tsai, Ruth Nussinov, Nurcan Tuncbag.

Here, we discover potential 'latent driver' mutations in cancer genomes. Latent drivers have low frequencies and minor observable translational potential. As such, to date they have escaped identification. Their discovery is important, since when paired in cis, latent driver mutations can drive cancer. Our comprehensive statistical analysis of the pan-cancer mutation profiles of ~60,000 tumor sequences from the TCGA and AACR-GENIE cohorts identifies significantly co-occurring potential latent drivers. We observe 155 same gene double mutations of which 140 individual components are cataloged as latent drivers. Evaluation of cell lines and patient-derived xenograft response data to drug treatment indicate that in certain genes double mutations may have a prominent role in increasing oncogenic activity, hence obtaining a better drug response, as in PIK3CA. Taken together, our comprehensive analyses indicate that same-gene double mutations are exceedingly rare phenomena but are a signature for some cancer types, e.g., breast, and lung cancers. The relative rarity of doublets can be explained by the likelihood of strong signals resulting in oncogene-induced senescence, and by doublets consisting of non-identical single residue components populating the background mutational load, thus not identified.

DOI: https://doi.org/10.1038/s42003-023-04519-5
Cancers (Basel). 2023 Feb 07. pii: 1060. [Epub ahead of print]15(4):

Pixelated Microfluidics for Drug Screening on Tumour Spheroids and Ex Vivo Microdissected Tumour Explants.

Dina Dorrigiv, Pierre-Alexandre Goyette, Amélie St-Georges-Robillard, Anne-Marie Mes-Masson, Thomas Gervais.

  Anticancer drugs have the lowest success rate of approval in drug development programs. Thus, preclinical assays that closely predict the clinical responses to drugs are of utmost importance in both clinical oncology and pharmaceutical research. 3D tumour models preserve the tumoral architecture and are cost- and time-efficient. However, the short-term longevity, limited throughput, and limitations of live imaging of these models have so far driven researchers towards less realistic tumour models such as monolayer cell cultures. Here, we present an open-space microfluidic drug screening platform that enables the formation, culture, and multiplexed delivery of several reagents to various 3D tumour models, namely cancer cell line spheroids and ex vivo primary tumour fragments. Our platform utilizes a microfluidic pixelated chemical display that creates isolated adjacent flow sub-units of reagents, which we refer to as fluidic 'pixels', over tumour models in a contact-free fashion. Up to nine different treatment conditions can be tested over 144 samples in a single experiment. We provide a proof-of-concept application by staining fixed and live tumour models with multiple cellular dyes. Furthermore, we demonstrate that the response of the tumour models to biological stimuli can be assessed using the platform. Upscaling the microfluidic platform to larger areas can lead to higher throughputs, and thus will have a significant impact on developing treatments for cancer.

Keywords:  3D tumour models; drug development; ex vivo tumour explants; open-space microfluidics; personalized medicine; preclinical assay; spheroids

DOI:  https://doi.org/10.3390/cancers15041060
Bioengineering (Basel). 2023 Feb 18. pii: 269. [Epub ahead of print]10(2):

Oscillatory ERK Signaling and Morphology Determine Heterogeneity of Breast Cancer Cell Chemotaxis via MEK-ERK and p38-MAPK Signaling Pathways.

Kenneth K Y Ho, Siddhartha Srivastava, Patrick C Kinnunen, Krishna Garikipati, Gary D Luker, Kathryn E Luker.

  Chemotaxis, regulated by oscillatory signals, drives critical processes in cancer metastasis. Crucial chemoattractant molecules in breast cancer, CXCL12 and EGF, drive the activation of ERK and Akt. Regulated by feedback and crosstalk mechanisms, oscillatory signals in ERK and Akt control resultant changes in cell morphology and chemotaxis. While commonly studied at the population scale, metastasis arises from small numbers of cells that successfully disseminate, underscoring the need to analyze processes that cancer cells use to connect oscillatory signaling to chemotaxis at single-cell resolution. Furthermore, little is known about how to successfully target fast-migrating cells to block metastasis. We investigated to what extent oscillatory networks in single cells associate with heterogeneous chemotactic responses and how targeted inhibitors block signaling processes in chemotaxis. We integrated live, single-cell imaging with time-dependent data processing to discover oscillatory signal processes defining heterogeneous chemotactic responses. We identified that short ERK and Akt waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, long ERK waves and the morphological changes regulated by MEK-ERK signaling, determine heterogeneous directed motion. This study indicates that treatments against chemotaxis in consider must interrupt oscillatory signaling.

Keywords:  CXCR4 signaling; cancer metastasis; chemotaxis; data-driven methods; heterogeneity; morphology; oscillation; single-cell imaging; system Identification

DOI:  https://doi.org/10.3390/bioengineering10020269
Nature. 2023 Feb 20.

Daily briefing: How artificial intelligence might change mathematics.

Flora Graham.

DOI: https://doi.org/10.1038/d41586-023-00522-2
Anal Methods. 2023 Feb 20.

An angled-shape tip-based strategy for highly sensitive proteomic profiling of a low number of cells.

Hongxian Zhao, Yongle Chen, Hang Li, Yangjun Zhang, Wanjun Zhang, Weijie Qin.

Profiling proteins plays an essential role in understanding the functions and dynamic networks in biological systems. Mass spectrometry-based proteomic analysis commonly requires multistep sample processing, which results in severe sample loss. Although the recently developed microproteomic strategies have substantially reduced sample loss via droplet microfluidic technology, specialized equipment and well-trained personnel are needed, which may limit their wide adoption. Here, we report an angled-shape tip-based strategy for rapid sample preparation and sensitive proteomic profiling of small cell populations (<1000 cells). The angled-shape tip provided a 'reactor' for the entire proteomic sample processing workflow, from cell capture and lysis to protein digestion, eliminating the sample transfer-induced protein loss. The angled-shape tip was surface-treated for anti-protein adsorption which further reduced the sample loss. Using this strategy, 1241 ± 38-4110 ± 37 protein groups and 4010 ± 700-34 879 ± 575 peptides were identified from 10-1000 HeLa cells with high quantification reproducibility in only 4.5 h sample processing time, which was superior to the reported methods and commercial kits, especially for <100 cells. This approach was easily accessible, straightforward to operate, and compatible with flow cytometry-based cell sorting. It showed great potential for in-depth proteomic profiling of rare cells (<1000 cells) in both basic biological research and clinical application.

DOI: https://doi.org/10.1039/d2ay01884e
bioRxiv. 2023 Feb 16. pii: 2023.02.15.528768. [Epub ahead of print]

Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.

Yuanhui Mao, Longfei Jia, Leiming Dong, Xin Erica Shu, Shu-Bing Qian.

A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation immediately from the start codon. The start codon-associated ribosome frameshifting (SCARF) stems from the slippage of ribosomes during the transition from initiation to elongation. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra unannotated from human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity by stabilizing initiating ribosomes. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF protects cells from starvation by enabling amino acid recycling and selective mRNA translation. Our findings illustrate a beneficial effect of translational "noise" in nutrient stress adaptation.

DOI: https://doi.org/10.1101/2023.02.15.528768
Cell Rep Methods. 2023 Jan 23. 3(1): 100392

Identifying key multifunctional components shared by critical cancer and normal liver pathways via SparseGMM.

Shaimaa Bakr, Kevin Brennan, Pritam Mukherjee, Josepmaria Argemi, Mikel Hernaez, Olivier Gevaert.

Despite the abundance of multimodal data, suitable statistical models that can improve our understanding of diseases with genetic underpinnings are challenging to develop. Here, we present SparseGMM, a statistical approach for gene regulatory network discovery. SparseGMM uses latent variable modeling with sparsity constraints to learn Gaussian mixtures from multiomic data. By combining coexpression patterns with a Bayesian framework, SparseGMM quantitatively measures confidence in regulators and uncertainty in target gene assignment by computing gene entropy. We apply SparseGMM to liver cancer and normal liver tissue data and evaluate discovered gene modules in an independent single-cell RNA sequencing (scRNA-seq) dataset. SparseGMM identifies PROCR as a regulator of angiogenesis and PDCD1LG2 and HNF4A as regulators of immune response and blood coagulation in cancer. Furthermore, we show that more genes have significantly higher entropy in cancer compared with normal liver. Among high-entropy genes are key multifunctional components shared by critical pathways, including p53 and estrogen signaling.

DOI: https://doi.org/10.1016/j.crmeth.2022.100392
Nat Commun. 2023 Feb 20. 14(1): 939

A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells.

Timothy K Turkalo, Antonio Maffia, Johannes J Schabort, Samuel G Regalado, Mital Bhakta, Marco Blanchette, Diana C J Spierings, Peter M Lansdorp, Dirk Hockemeyer.

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.

DOI: https://doi.org/10.1038/s41467-023-36294-6
PLoS Comput Biol. 2023 Feb 21. 19(2): e1010888

Kinome inhibition states and multiomics data enable prediction of cell viability in diverse cancer types.

Matthew E Berginski, Chinmaya U Joisa, Brian T Golitz, Shawn M Gomez.

Protein kinases play a vital role in a wide range of cellular processes, and compounds that inhibit kinase activity emerging as a primary focus for targeted therapy development, especially in cancer. Consequently, efforts to characterize the behavior of kinases in response to inhibitor treatment, as well as downstream cellular responses, have been performed at increasingly large scales. Previous work with smaller datasets have used baseline profiling of cell lines and limited kinome profiling data to attempt to predict small molecule effects on cell viability, but these efforts did not use multi-dose kinase profiles and achieved low accuracy with very limited external validation. This work focuses on two large-scale primary data types, kinase inhibitor profiles and gene expression, to predict the results of cell viability screening. We describe the process by which we combined these data sets, examined their properties in relation to cell viability and finally developed a set of computational models that achieve a reasonably high prediction accuracy (R2 of 0.78 and RMSE of 0.154). Using these models, we identified a set of kinases, several of which are understudied, that are strongly influential in the cell viability prediction models. In addition, we also tested to see if a wider range of multiomics data sets could improve the model results and found that proteomic kinase inhibitor profiles were the single most informative data type. Finally, we validated a small subset of the model predictions in several triple-negative and HER2 positive breast cancer cell lines demonstrating that the model performs well with compounds and cell lines that were not included in the training data set. Overall, this result demonstrates that generic knowledge of the kinome is predictive of very specific cell phenotypes, and has the potential to be integrated into targeted therapy development pipelines.

DOI: https://doi.org/10.1371/journal.pcbi.1010888
Nat Commun. 2023 Feb 23. 14(1): 998

A conversation about building a support network in science.

DOI: https://doi.org/10.1038/s41467-023-36599-6
Cell Syst. 2023 Feb 15. pii: S2405-4712(23)00031-5. [Epub ahead of print]

Leading edge competition promotes context-dependent responses to receptor inputs to resolve directional dilemmas in neutrophil migration.

Amalia Hadjitheodorou, George R R Bell, Felix Ellett, Daniel Irimia, Robert Tibshirani, Sean R Collins, Julie A Theriot.

  Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.

Keywords:  Cdc42; cytoskeleton; microfluidics; motility; neutrophils; optogenetics; polarity; statistical learning

DOI:  https://doi.org/10.1016/j.cels.2023.02.001
Stem Cell Reports. 2023 Feb 15. pii: S2213-6711(23)00011-5. [Epub ahead of print]

In silico discovery of small molecules for efficient stem cell differentiation into definitive endoderm.

Gherman Novakovsky, Shugo Sasaki, Oriol Fornes, Meltem E Omur, Helen Huang, Carmen L Bayly, Dahai Zhang, Nathaniel Lim, Artem Cherkasov, Paul Pavlidis, Sara Mostafavi, Francis C Lynn, Wyeth W Wasserman.

  Improving methods for human embryonic stem cell differentiation represents a challenge in modern regenerative medicine research. Using drug repurposing approaches, we discover small molecules that regulate the formation of definitive endoderm. Among them are inhibitors of known processes involved in endoderm differentiation (mTOR, PI3K, and JNK pathways) and a new compound, with an unknown mechanism of action, capable of inducing endoderm formation in the absence of growth factors in the media. Optimization of the classical protocol by inclusion of this compound achieves the same differentiation efficiency with a 90% cost reduction. The presented in silico procedure for candidate molecule selection has broad potential for improving stem cell differentiation protocols.

Keywords:  bioinformatics; definitive endoderm; differentiation; drug repurposing; growth factor; pathway analysis; stem cell; transcription

DOI:  https://doi.org/10.1016/j.stemcr.2023.01.008
bioRxiv. 2023 Feb 16. pii: 2023.02.15.528736. [Epub ahead of print]

Network-based elucidation of colon cancer drug resistance by phosphoproteomic time-series analysis.

George Rosenberger, Wenxue Li, Mikko Turunen, Jing He, Prem S Subramaniam, Sergey Pampou, Aaron T Griffin, Charles Karan, Patrick Kerwin, Diana Murray, Barry Honig, Yansheng Liu, Andrea Califano.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies, over the past decade, we developed VESPA-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases.

DOI: https://doi.org/10.1101/2023.02.15.528736
Elife. 2023 Feb 21. pii: e81048. [Epub ahead of print]12

Nuclear export inhibition jumbles epithelial-mesenchymal states and gives rise to migratory disorder in healthy epithelia.

Carly M Krull, Haiyi Li, Amit Pathak.

  Dynamic nucleocytoplasmic transport of E-M factors regulates cellular E-M states; yet, it remains unknown how simultaneously trapping these factors affects epithelia at the macroscale. To explore this question, we performed nuclear export inhibition (NEI) via leptomycin B and Selinexor treatment, which biases nuclear localization of CRM1-associated E-M factors. We examined changes in collective cellular phenotypes across a range of substrate stiffnesses. Following NEI, soft substrates elevate collective migration of MCF10A cells for up to 24 hr, while stiffer substrates reduce migration at all time points. Our results suggest that NEI disrupts migration through competition between intercellular adhesions and mechanoactivation, generally causing loss of cell-cell coordination. Specifically, across substrate stiffnesses, NEI fosters an atypical E-M state wherein MCF10A cells become both more epithelial and more mesenchymal. We observe that NEI fosters a range of these concurrent phenotypes, from more epithelial shYAP MCF10A cells to more mesenchymal MDCK II cells. α-Catenin emerges as a potential link between E-M states, where it maintains normal levels of intercellular adhesion and transmits mechanoactive characteristics to collective behavior. Ultimately, to accommodate the concurrent states observed here, we propose an expanded E-M model, which may help further understand fundamental biological phenomena and inform pathological treatments.

Keywords:  EMT; collective cell migration; epithelial cells; epithelial–mesenchymal transition; mechanobiology; nuclear export; nucleocytoplasmic transport; physics of living systems

DOI:  https://doi.org/10.7554/eLife.81048
Cell Chem Biol. 2023 Feb 17. pii: S2451-9456(23)00033-8. [Epub ahead of print]

Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction.

Jun Yang, Peihao Chen, Yu Cao, Shanshan Liu, Wei Wang, Lin Li, Jiaojiao Li, Zhaodi Jiang, Yan Ma, She Chen, Sanduo Zheng, Xiangbing Qi, Hui Jiang.

  Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.

Keywords:  DRP1 inhibitor; MFF; MIDI; MiD49/51; OPA1; mitochondrial dynamics; mitochondrial fission; mitofusin

DOI:  https://doi.org/10.1016/j.chembiol.2023.02.002
Commun Biol. 2023 Feb 20. 6(1): 203

Endogenous DAF-16 spatiotemporal activity quantitatively predicts lifespan extension induced by dietary restriction.

Javier Huayta, Joseph P Crapster, Adriana San-Miguel.

In many organisms, dietary restriction (DR) leads to lifespan extension through the activation of cell protection and pro-longevity gene expression programs. In the nematode C. elegans, the DAF-16 transcription factor is a key aging regulator that governs the Insulin/IGF-1 signaling pathway and undergoes translocation from the cytoplasm to the nucleus of cells when animals are exposed to food limitation. However, how large is the influence of DR on DAF-16 activity, and its subsequent impact on lifespan has not been quantitatively determined. In this work, we assess the endogenous activity of DAF-16 under various DR regimes by coupling CRISPR/Cas9-enabled fluorescent tagging of DAF-16 with quantitative image analysis and machine learning. Our results indicate that DR regimes induce strong endogenous DAF-16 activity, although DAF-16 is less responsive in aged individuals. DAF-16 activity is in turn a robust predictor of mean lifespan in C. elegans, accounting for 78% of its variability under DR. Analysis of tissue-specific expression aided by a machine learning tissue classifier reveals that, under DR, the largest contribution to DAF-16 nuclear intensity originates from the intestine and neurons. DR also drives DAF-16 activity in unexpected locations such as the germline and intestinal nucleoli.

DOI: https://doi.org/10.1038/s42003-023-04562-2
Front Neurosci. 2022 ;16 972201

Large-scale organoid study suggests effects of trisomy 21 on early fetal neurodevelopment are more subtle than variability between isogenic lines and experiments.

Jan T Czerminski, Oliver D King, Jeanne B Lawrence.

  This study examines cortical organoids generated from a panel of isogenic trisomic and disomic iPSC lines (subclones) as a model of early fetal brain development in Down syndrome (DS). An initial experiment comparing organoids from one trisomic and one disomic line showed many genome-wide transcriptomic differences and modest differences in cell-type proportions, suggesting there may be a neurodevelopmental phenotype that is due to trisomy of chr21. To better control for multiple sources of variation, we undertook a highly robust study of ∼1,200 organoids using an expanded panel of six all-isogenic lines, three disomic, and three trisomic. The power of this experimental design was indicated by strong detection of the ∼1.5-fold difference in chr21 genes. However, the numerous expression differences in non-chr21 genes seen in the smaller experiment fell away, and the differences in cell-type representation between lines did not correlate with trisomy 21. Results suggest that the initial smaller experiment picked up differences between small organoid samples and individual isogenic lines, which "averaged out" in the larger panel of isogenic lines. Our results indicate that even when organoid and batch variability are better controlled for, variation between isogenic cell lines (even subclones) may obscure, or be conflated with, subtle neurodevelopmental phenotypes that may be present in ∼2nd trimester DS brain development. Interestingly, despite this variability between organoid batches and lines, and the "fetal stage" of these organoids, an increase in secreted Aβ40 peptide levels-an Alzheimer-related cellular phenotype-was more strongly associated with trisomy 21 status than were neurodevelopmental shifts in cell-type composition.

Keywords:  Alzheimer’s disease; Down syndrome; cerebral organoids; iPS cells; neurodevelopment

DOI:  https://doi.org/10.3389/fnins.2022.972201