bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒07‒23
twenty-two papers selected by
Ralitsa Radostinova Madsen
MRC-PPU
  1. Characterization of Lipid Alterations by Oncogenic PIK3CA Mutations Using Untargeted Lipidomics in Breast Cancer.
  2. The mechanisms of class 1A PI3K and Wnt/β-catenin coupled signaling in breast cancer.
  3. PI4KA and PIKfyve: Essential phosphoinositide signaling enzymes involved in myriad human diseases.
  4. A critical evaluation of protein kinase regulation by activation loop autophosphorylation.
  5. Simufilam suppresses overactive mTOR and restores its sensitivity to insulin in Alzheimer's disease patient lymphocytes.
  6. Distinct shared and compartment-enriched oncogenic networks drive primary versus metastatic breast cancer.
  7. RAGE inhibition blunts insulin-induced oncogenic signals in breast cancer.
  8. Targeting PI3Kα increases the efficacy of anti-PD-1 antibody in cervical cancer.
  9. Biphasic JNK-Erk signaling separates the induction and maintenance of cell senescence after DNA damage induced by topoisomerase II inhibition.
  10. Organ Mapping Antibody Panels: a community resource for standardized multiplexed tissue imaging.
  11. A focus on single-cell omics.
  12. Identification of transcriptional programs using dense vector representations defined by mutual information with GeneVector.
  13. Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape.
  14. EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.
  15. Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols.
  16. Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.
  17. Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP).
  18. More than bad luck: Cancer and aging are linked to replication-driven changes to the epigenome.
  19. Liver insulinization as a driver of triglyceride dysmetabolism.
  20. Bifunctional small molecules that induce nuclear localization and targeted transcriptional regulation.
  21. Orphan quality control shapes network dynamics and gene expression.
  22. Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption.
  1. OMICS. 2023 Jul;27(7): 327-335
    Characterization of Lipid Alterations by Oncogenic PIK3CA Mutations Using Untargeted Lipidomics in Breast Cancer.
    Jae Hun Jung, Da-Qing Yang, Hongming Song, Xiangyu Wang, Xinyan Wu, Kwang Pyo Kim, Akhilesh Pandey, Seul Kee Byeon.
      Lipids play crucial biological roles in health and disease, including in cancers. The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pivotal promoter of cell growth and proliferation in various types of cancer. The somatic mutations in PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, are frequently present in cancer cells, including breast cancer. Although the most prominent mutants, represented by single amino acid substitutions in the helical domain in exon 9 (E545K) and the kinase domain in exon 20 (H1047R) are known to cause a gain of PI3K function, activate AKT signaling and induce oncogenic transformation, the effect of these mutations on cellular lipid profiles has not been studied. We carried out untargeted lipidomics using liquid chromatography-tandem mass spectrometry to detect the lipid alterations in mammary gland epithelial MCF10A cells with isogenic knockin of these mutations. A total of 536 species of lipids were analyzed. We found that the levels of monosialogangliosides, signaling molecules known to enhance cell motility through PI3K/AKT pathway, were significantly higher in both mutants. In addition, triglycerides and ceramides, lipid molecules known to be involved in promoting lipid droplet production, cancer cell migration and invasion, were increased, whereas lysophosphatidylcholines and phosphatidylcholines that are known to inhibit cancer cell motility were decreased in both mutants. Our results provide novel insights into a potential link between altered lipid profile and carcinogenesis caused by the PIK3CA hotspot mutations. In addition, we suggest untargeted lipidomics offers prospects for precision/personalized medicine by unpacking new molecular substrates of cancer biology.
    Keywords:  biomarkers; cancer; integrative biology; lipidomics; mutations; personalized medicine
    DOI:  https://doi.org/10.1089/omi.2023.0076
  2. Biochem Soc Trans. 2023 Jul 20. pii: BST20220866. [Epub ahead of print]
    The mechanisms of class 1A PI3K and Wnt/β-catenin coupled signaling in breast cancer.
    Samuel J Rodgers, Christina A Mitchell, Lisa M Ooms.
      The class IA PI3K signaling pathway is activated by growth factor stimulation and regulates a signaling cascade that promotes diverse events including cell growth, proliferation, migration and metabolism. PI3K signaling is one of the most commonly hyperactivated pathways in breast cancer, leading to increased tumor growth and progression. PI3K hyperactivation occurs via a number of genetic and epigenetic mechanisms including mutation or amplification of PIK3CA, the gene encoding the p110α subunit of PI3Kα, as well as via dysregulation of the upstream growth factor receptors or downstream signaling effectors. Over the past decade, extensive efforts to develop therapeutics that suppress oncogenic PI3K signaling have been undertaken. Although FDA-approved PI3K inhibitors are now emerging, their clinical success remains limited due to adverse effects and negative feedback mechanisms which contribute to their reduced efficacy. There is an emerging body of evidence demonstrating crosstalk between the PI3K and Wnt/β-catenin pathways in breast cancer. However, PI3K exhibits opposing effects on Wnt/β-catenin signaling in distinct tumor subsets, whereby PI3K promotes Wnt/β-catenin activation in ER+ cancers, but paradoxically suppresses this pathway in ER- breast cancers. This review discusses the molecular mechanisms for PI3K-Wnt crosstalk in breast cancer, and how Wnt-targeted therapies have the potential to contribute to treatment regimens for breast cancers with PI3K dysregulation.
    Keywords:  INPP4B; Wnt/β-catenin; breast cancer; phosphoinositide; phosphoinositide 3-kinase
    DOI:  https://doi.org/10.1042/BST20220866
  3. Curr Opin Cell Biol. 2023 Jul 13. pii: S0955-0674(23)00056-X. [Epub ahead of print]83 102207
    PI4KA and PIKfyve: Essential phosphoinositide signaling enzymes involved in myriad human diseases.
    Isobel Barlow-Busch, Alexandria L Shaw, John E Burke.
      Lipid phosphoinositides are master regulators of multiple cellular functions. Misregulation of the activity of the lipid kinases that generate phosphoinositides is causative of human diseases, including cancer, neurodegeneration, developmental disorders, immunodeficiencies, and inflammatory disease. This review will present a summary of recent discoveries on the roles of two phosphoinositide kinases (PI4KA and PIKfyve), which have emerged as targets for therapeutic intervention. Phosphatidylinositol 4-kinase alpha (PI4KA) generates PI4P at the plasma membrane and PIKfyve generates PI(3,5)P2 at endo-lysosomal membranes. Both of these enzymes exist as multi-protein mega complexes that are under myriad levels of regulation. Human disease can be caused by either loss or gain-of-function of these complexes, so understanding how they are regulated will be essential in the design of therapeutics. We will summarize insight into how these enzymes are regulated by their protein-binding partners, with a major focus on the unanswered questions of how their activity is controlled.
    DOI:  https://doi.org/10.1016/j.ceb.2023.102207
  4. Elife. 2023 07 20. pii: e88210. [Epub ahead of print]12
    A critical evaluation of protein kinase regulation by activation loop autophosphorylation.
    Ronja Reinhardt, Thomas A Leonard.
      Phosphorylation of proteins is a ubiquitous mechanism of regulating their function, localization, or activity. Protein kinases, enzymes that use ATP to phosphorylate protein substrates are, therefore, powerful signal transducers in eukaryotic cells. The mechanism of phosphoryl-transfer is universally conserved among protein kinases, which necessitates the tight regulation of kinase activity for the orchestration of cellular processes with high spatial and temporal fidelity. In response to a stimulus, many kinases enhance their own activity by autophosphorylating a conserved amino acid in their activation loop, but precisely how this reaction is performed is controversial. Classically, kinases that autophosphorylate their activation loop are thought to perform the reaction in trans, mediated by transient dimerization of their kinase domains. However, motivated by the recently discovered regulation mechanism of activation loop cis-autophosphorylation by a kinase that is autoinhibited in trans, we here review the various mechanisms of autoregulation that have been proposed. We provide a framework for critically evaluating biochemical, kinetic, and structural evidence for protein kinase dimerization and autophosphorylation, and share some thoughts on the implications of these mechanisms within physiological signaling networks.
    Keywords:  activation; allostery; biochemistry; chemical biology; dimerization; inhibition; kinase; molecular biophysics; phosphorylation; structural biology
    DOI:  https://doi.org/10.7554/eLife.88210
  5. Front Aging. 2023 ;4 1175601
    Simufilam suppresses overactive mTOR and restores its sensitivity to insulin in Alzheimer's disease patient lymphocytes.
    Hoau-Yan Wang, Zhe Pei, Kuo-Chieh Lee, Boris Nikolov, Tamara Doehner, John Puente, Nadav Friedmann, Lindsay H Burns.
      Introduction: Implicated in both aging and Alzheimer's disease (AD), mammalian target of rapamycin (mTOR) is overactive in AD brain and lymphocytes. Stimulated by growth factors such as insulin, mTOR monitors cell health and nutrient needs. A small molecule oral drug candidate for AD, simufilam targets an altered conformation of the scaffolding protein filamin A (FLNA) found in AD brain and lymphocytes that induces aberrant FLNA interactions leading to AD neuropathology. Simufilam restores FLNA's normal shape to disrupt its AD-associated protein interactions. Methods: We measured mTOR and its response to insulin in lymphocytes of AD patients before and after oral simufilam compared to healthy control lymphocytes. Results: mTOR was overactive and its response to insulin reduced in lymphocytes from AD versus healthy control subjects, illustrating another aspect of insulin resistance in AD. After oral simufilam, lymphocytes showed normalized basal mTOR activity and improved insulin-evoked mTOR activation in mTOR complex 1, complex 2, and upstream and downstream signaling components (Akt, p70S6K and phosphorylated Rictor). Suggesting mechanism, we showed that FLNA interacts with the insulin receptor until dissociation by insulin, but this linkage was elevated and its dissociation impaired in AD lymphocytes. Simufilam improved the insulin-mediated dissociation. Additionally, FLNA's interaction with Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), a negative regulator of mTOR, was reduced in AD lymphocytes and improved by simufilam. Discussion: Reducing mTOR's basal overactivity and its resistance to insulin represents another mechanism of simufilam to counteract aging and AD pathology. Simufilam is currently in Phase 3 clinical trials for AD dementia.
    Keywords:  Alzheimer’s disease; PTEN; aging; filamin A; insulin resistance; mTOR
    DOI:  https://doi.org/10.3389/fragi.2023.1175601
  6. Nat Commun. 2023 07 18. 14(1): 4313
    Distinct shared and compartment-enriched oncogenic networks drive primary versus metastatic breast cancer.
    Zhe Jiang, YoungJun Ju, Amjad Ali, Philip E D Chung, Patryk Skowron, Dong-Yu Wang, Mariusz Shrestha, Huiqin Li, Jeff C Liu, Ioulia Vorobieva, Ronak Ghanbari-Azarnier, Ethel Mwewa, Marianne Koritzinsky, Yaacov Ben-David, James R Woodgett, Charles M Perou, Adam Dupuy, Gary D Bader, Sean E Egan, Michael D Taylor, Eldad Zacksenhaus.
      Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFβ and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.
    DOI:  https://doi.org/10.1038/s41467-023-39935-y
  7. Breast Cancer Res. 2023 07 17. 25(1): 84
    RAGE inhibition blunts insulin-induced oncogenic signals in breast cancer.
    M G Muoio, M Pellegrino, V Rapicavoli, M Talia, G Scavo, V Sergi, V Vella, S Pettinato, M G Galasso, R Lappano, D Scordamaglia, F Cirillo, A Pulvirenti, D C Rigiracciolo, M Maggiolini, A Belfiore, E M De Francesco.
      The receptor for advanced glycation end products (RAGE) is implicated in diabetes and obesity complications, as well as in breast cancer (BC). Herein, we evaluated whether RAGE contributes to the oncogenic actions of Insulin, which plays a key role in BC progression particularly in obese and diabetic patients. Analysis of the publicly available METABRIC study, which collects gene expression and clinical data from a large cohort (n = 1904) of BC patients, revealed that RAGE and the Insulin Receptor (IR) are co-expressed and associated with negative prognostic parameters. In MCF-7, ZR75 and 4T1 BC cells, as well as in patient-derived Cancer-Associated Fibroblasts, the pharmacological inhibition of RAGE as well as its genetic depletion interfered with Insulin-induced activation of the oncogenic pathway IR/IRS1/AKT/CD1. Mechanistically, IR and RAGE directly interacted upon Insulin stimulation, as shown by in situ proximity ligation assays and coimmunoprecipitation studies. Of note, RAGE inhibition halted the activation of both IR and insulin like growth factor 1 receptor (IGF-1R), as demonstrated in MCF-7 cells KO for the IR and the IGF-1R gene via CRISPR-cas9 technology. An unbiased label-free proteomic analysis uncovered proteins and predicted pathways affected by RAGE inhibition in Insulin-stimulated BC cells. Biologically, RAGE inhibition reduced cell proliferation, migration, and patient-derived mammosphere formation triggered by Insulin. In vivo, the pharmacological inhibition of RAGE halted Insulin-induced tumor growth, without affecting blood glucose homeostasis. Together, our findings suggest that targeting RAGE may represent an appealing opportunity to blunt Insulin-induced oncogenic signaling in BC.
    Keywords:  Breast cancer; CAFs; Insulin; Insulin receptor; RAGE
    DOI:  https://doi.org/10.1186/s13058-023-01686-5
  8. Immunology. 2023 Jul 19.
    Targeting PI3Kα increases the efficacy of anti-PD-1 antibody in cervical cancer.
    Wei Jiang, Xueyan Ouyang, Chunyan Li, Yixiu Long, Wei Chen, Zhaodong Ji, Xuxia Shen, Libing Xiang, Huijuan Yang.
      Targeting programmed death 1(PD-1) has been approved for relapsed cervical cancer with unsatisfactory clinical efficacy. This study aims to analyse the impact of PI3K pathway activation on tumour immune microenvironment and evaluates the immune sensitization effect by PI3K inhibition in cervical cancer. The effect of PIK3CA mutation on PD-L1 expression and CD8+ T cells differentiation was determined in cervical cancer tissues. Luciferase and ChIP-qPCR/PCR assays were used to determine the transcriptional regulation of PD-L1 by PIK3CA-E545K. The effects of PI3K inhibitor treatment on immune environment in vitro and in vivo were evaluated by RNA sequencing (RNA-seq) and flow cytometry. The efficacy of PI3K inhibitor and anti-PD-1 therapy was assessed in cell-derived xenografts (CDX) and patients-derived xenografts (PDX). PD-L1 overexpression is more frequently observed in elder women with squamous cervical carcinoma. It predicts longer progress-free survival and overall survival. PIK3CA mutation results in increased mRNA and protein levels of PD-L1, the repression of CD8+ T cell differentiation in cervical cancer. Here, we report a case that continuous pembrolizumab monotherapy treatment induced complete remission of a recurrent cervical cancer patient with systemic metastasis and PIK3CA-E545K mutation, implying that PIK3CA mutation is potentially a biomarker for pembrolizumab treatment in cervical cancer. Specifically, this mutation promotes the expression of PD-L1 by upregulating the transcription factor IRF1. PI3Kα-specific inhibitor markedly activates immune microenvironment by regulating the PD-1/L1-related pathways and promoting CD8+ T cell differentiation and proliferation in Caski-CDXs with PIK3CA-E545K mutation. PI3Kα inhibitor significantly enhances the anti-tumour efficacy of PD-1 blockade in CDXs and PDXs. PIK3CA mutations may predict the response of cervical cancer to PD-1 blockade. The efficacy of PI3Kα inhibitors combined with PD-1 antibodies is promising in cervical cancer and warrants additional clinical and mechanistic investigations.
    Keywords:  PD-1/L1; PIK3CA mutation; cervical cancer; immune checkpoint blockade
    DOI:  https://doi.org/10.1111/imm.13682
  9. Cell Syst. 2023 Jul 19. pii: S2405-4712(23)00158-8. [Epub ahead of print]14(7): 582-604.e10
    Biphasic JNK-Erk signaling separates the induction and maintenance of cell senescence after DNA damage induced by topoisomerase II inhibition.
    Tatiana S Netterfield, Gerard J Ostheimer, Andrea R Tentner, Brian A Joughin, Alexandra M Dakoyannis, Charvi D Sharma, Peter K Sorger, Kevin A Janes, Douglas A Lauffenburger, Michael B Yaffe.
      Genotoxic stress in mammalian cells, including those caused by anti-cancer chemotherapy, can induce temporary cell-cycle arrest, DNA damage-induced senescence (DDIS), or apoptotic cell death. Despite obvious clinical importance, it is unclear how the signals emerging from DNA damage are integrated together with other cellular signaling pathways monitoring the cell's environment and/or internal state to control different cell fates. Using single-cell-based signaling measurements combined with tensor partial least square regression (t-PLSR)/principal component analysis (PCA) analysis, we show that JNK and Erk MAPK signaling regulates the initiation of cell senescence through the transcription factor AP-1 at early times after doxorubicin-induced DNA damage and the senescence-associated secretory phenotype (SASP) at late times after damage. These results identify temporally distinct roles for signaling pathways beyond the classic DNA damage response (DDR) that control the cell senescence decision and modulate the tumor microenvironment and reveal fundamental similarities between signaling pathways responsible for oncogene-induced senescence (OIS) and senescence caused by topoisomerase II inhibition. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  AP-1; DNA damage; Erk; JNK; MAPK; c-Jun; cancer therapy; senescence; senescence-associated secretory phenotype; tensor PLSR; topoisomerase
    DOI:  https://doi.org/10.1016/j.cels.2023.06.005
  10. Nat Methods. 2023 Jul 19.
    Organ Mapping Antibody Panels: a community resource for standardized multiplexed tissue imaging.
    Ellen M Quardokus, Diane C Saunders, Elizabeth McDonough, John W Hickey, Christopher Werlein, Christine Surrette, Presha Rajbhandari, Anna Martinez Casals, Hua Tian, Lisa Lowery, Elizabeth K Neumann, Frida Björklund, Taruna V Neelakantan, Josh Croteau, Anne E Wiblin, Jeremy Fisher, April J Livengood, Karen G Dowell, Jonathan C Silverstein, Jeffrey M Spraggins, Gloria S Pryhuber, Gail Deutsch, Fiona Ginty, Garry P Nolan, Simon Melov, Danny Jonigk, Michael A Caldwell, Ioannis S Vlachos, Werner Muller, Nils Gehlenborg, Brent R Stockwell, Emma Lundberg, Michael P Snyder, Ronald N Germain, Jeannie M Camarillo, Neil L Kelleher, Katy Börner, Andrea J Radtke.
      Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.
    DOI:  https://doi.org/10.1038/s41592-023-01846-7
  11. Nat Rev Genet. 2023 Aug;24(8): 485
    A focus on single-cell omics.
      
    DOI:  https://doi.org/10.1038/s41576-023-00628-3
  12. Nat Commun. 2023 Jul 20. 14(1): 4400
    Identification of transcriptional programs using dense vector representations defined by mutual information with GeneVector.
    Nicholas Ceglia, Zachary Sethna, Samuel S Freeman, Florian Uhlitz, Viktoria Bojilova, Nicole Rusk, Bharat Burman, Andrew Chow, Sohrab Salehi, Farhia Kabeer, Samuel Aparicio, Benjamin D Greenbaum, Sohrab P Shah, Andrew McPherson.
      Deciphering individual cell phenotypes from cell-specific transcriptional processes requires high dimensional single cell RNA sequencing. However, current dimensionality reduction methods aggregate sparse gene information across cells, without directly measuring the relationships that exist between genes. By performing dimensionality reduction with respect to gene co-expression, low-dimensional features can model these gene-specific relationships and leverage shared signal to overcome sparsity. We describe GeneVector, a scalable framework for dimensionality reduction implemented as a vector space model using mutual information between gene expression. Unlike other methods, including principal component analysis and variational autoencoders, GeneVector uses latent space arithmetic in a lower dimensional gene embedding to identify transcriptional programs and classify cell types. In this work, we show in four single cell RNA-seq datasets that GeneVector was able to capture phenotype-specific pathways, perform batch effect correction, interactively annotate cell types, and identify pathway variation with treatment over time.
    DOI:  https://doi.org/10.1038/s41467-023-39985-2
  13. Cell Syst. 2023 Jul 19. pii: S2405-4712(23)00154-0. [Epub ahead of print]14(7): 551-562.e5
    Optogenetic control of the integrated stress response reveals proportional encoding and the stress memory landscape.
    Taivan Batjargal, Francesca Zappa, Ryan J Grant, Robert A Piscopio, Alex Chialastri, Siddharth S Dey, Diego Acosta-Alvear, Maxwell Z Wilson.
      The integrated stress response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered a photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics, we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR, which demonstrated the dependence of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  Cryptochrome 2; RNA-seq; cellular resilience; integrated stress response; optogenetics; stress memory landscape; systems biology
    DOI:  https://doi.org/10.1016/j.cels.2023.06.001
  14. Circ Res. 2023 Jul 21.
    EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.
    Sila Algül, Maike Schuldt, Emmy Manders, Valentijn Jansen, Saskia Schlossarek, Richard de Goeij-de Haas, Alex A Henneman, Sander R Piersma, Connie R Jimenez, Michelle Michels, Lucie Carrier, Michiel Helmes, Jolanda van der Velden, Diederik W D Kuster.
      BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM.METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model.
    RESULTS: Screening of 157 kinase inhibitors in wild-type (n=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (n=24 HCM and n=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (n=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes.
    CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.
    Keywords:  animals; heart failure; mice; myocytes, cardiac; stroke volume
    DOI:  https://doi.org/10.1161/CIRCRESAHA.122.322133
  15. Stem Cell Reports. 2023 Jul 04. pii: S2213-6711(23)00237-0. [Epub ahead of print]
    Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols.
    Emily K W Lo, Jeremy J Velazquez, Da Peng, Chulan Kwon, Mo R Ebrahimkhani, Patrick Cahan.
      Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.
    Keywords:  Cardiomyocytes; Cell Engineering; Computational Biology; Directed Differentiation; Heart; Hepatocytes; Liver; Organoids; Transcriptomics; Transdifferentiation
    DOI:  https://doi.org/10.1016/j.stemcr.2023.06.008
  16. Nat Methods. 2023 Jul 20.
    Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.
    Stephan Riesenberg, Philipp Kanis, Dominik Macak, Damian Wollny, Dorothee Düsterhöft, Johannes Kowalewski, Nelly Helmbrecht, Tomislav Maricic, Svante Pääbo.
      Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.
    DOI:  https://doi.org/10.1038/s41592-023-01949-1
  17. Nat Cell Biol. 2023 Jul 19.
    Advances and prospects for the Human BioMolecular Atlas Program (HuBMAP).
    HuBMAP Consortium
      The Human BioMolecular Atlas Program (HuBMAP) aims to create a multi-scale spatial atlas of the healthy human body at single-cell resolution by applying advanced technologies and disseminating resources to the community. As the HuBMAP moves past its first phase, creating ontologies, protocols and pipelines, this Perspective introduces the production phase: the generation of reference spatial maps of functional tissue units across many organs from diverse populations and the creation of mapping tools and infrastructure to advance biomedical research.
    DOI:  https://doi.org/10.1038/s41556-023-01194-w
  18. Sci Adv. 2023 Jul 21. 9(29): eadf4163
    More than bad luck: Cancer and aging are linked to replication-driven changes to the epigenome.
    Christopher J Minteer, Kyra Thrush, John Gonzalez, Peter Niimi, Mariya Rozenblit, Joel Rozowsky, Jason Liu, Mor Frank, Thomas McCabe, Albert T Higgins-Chen, Erin Hofstatter, Lajos Pusztai, Kenneth Beckman, Mark Gerstein, Morgan E Levine.
      Aging is a leading risk factor for cancer. While it is proposed that age-related accumulation of somatic mutations drives this relationship, it is likely not the full story. We show that aging and cancer share a common epigenetic replication signature, which we modeled using DNA methylation from extensively passaged immortalized human cells in vitro and tested on clinical tissues. This signature, termed CellDRIFT, increased with age across multiple tissues, distinguished tumor from normal tissue, was escalated in normal breast tissue from cancer patients, and was transiently reset upon reprogramming. In addition, within-person tissue differences were correlated with predicted lifetime tissue-specific stem cell divisions and tissue-specific cancer risk. Our findings suggest that age-related replication may drive epigenetic changes in cells and could push them toward a more tumorigenic state.
    DOI:  https://doi.org/10.1126/sciadv.adf4163
  19. Nat Metab. 2023 Jul 17.
    Liver insulinization as a driver of triglyceride dysmetabolism.
    Joshua R Cook, Meredith A Hawkins, Utpal B Pajvani.
      Metabolic dysfunction-associated fatty liver disease (MAFLD) is an increasingly prevalent fellow traveller with the insulin resistance that underlies type 2 diabetes mellitus. However, the mechanistic connection between MAFLD and impaired insulin action remains unclear. In this Perspective, we review data from humans to elucidate insulin's aetiological role in MAFLD. We focus particularly on the relative preservation of insulin's stimulation of triglyceride (TG) biosynthesis despite its waning ability to curb hepatic glucose production (HGP). To explain this apparent 'selective insulin resistance', we propose that hepatocellular processes that lead to TG accumulation require less insulin signal transduction, or 'insulinization,' than do those that regulate HGP. As such, mounting hyperinsulinaemia that barely compensates for aberrant HGP in insulin-resistant states more than suffices to maintain hepatic TG biosynthesis. Thus, even modestly elevated or context-inappropriate insulin levels, when sustained day and night within a heavily pro-lipogenic metabolic milieu, may translate into substantial cumulative TG biosynthesis in the insulin-resistant state.
    DOI:  https://doi.org/10.1038/s42255-023-00843-6
  20. bioRxiv. 2023 Jul 07. pii: 2023.07.07.548101. [Epub ahead of print]
    Bifunctional small molecules that induce nuclear localization and targeted transcriptional regulation.
    William J Gibson, Ananthan Sadagopan, Veronika M Shoba, Amit Choudhary, Matthew Meyerson, Stuart L Schreiber.
      The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules, with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargoes into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CA E545K ), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemical-induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry with significant implications for disease therapy.
    DOI:  https://doi.org/10.1101/2023.07.07.548101
  21. Cell. 2023 Jul 14. pii: S0092-8674(23)00691-8. [Epub ahead of print]
    Orphan quality control shapes network dynamics and gene expression.
    Kevin G Mark, SriDurgaDevi Kolla, Jacob D Aguirre, Danielle M Garshott, Stefan Schmitt, Diane L Haakonsen, Christina Xu, Lukas Kater, Georg Kempf, Brenda Martínez-González, David Akopian, Stephanie K See, Nicolas H Thomä, Michael Rapé.
      All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.
    Keywords:  MCRS1; MYC; UBR5; branched ubiquitin chain; orphan quality control; proteasome; stem cell; transcription factor; ubiquitin
    DOI:  https://doi.org/10.1016/j.cell.2023.06.015
  22. Circ Res. 2023 Jul 18.
    Chylomicrons Regulate Lacteal Permeability and Intestinal Lipid Absorption.
    Georgia Zarkada, Xun Chen, Xuetong Zhou, Martin Lange, Lei Zeng, Wenyu Lv, Xuan Zhang, Yunhua Li, Weibin Zhou, Keli Liu, Dongying Chen, Nicolas Ricard, James Liao, Young-Bum Kim, Rui Benedito, Lena Claesson-Welsh, Kari Alitalo, Michael Simons, Rong Ju, Xuri Li, Anne Eichmann, Feng Zhang.
      BACKGROUND: Lymphatic vessels are responsible for tissue drainage, and their malfunction is associated with chronic diseases. Lymph uptake occurs via specialized open cell-cell junctions between capillary lymphatic endothelial cells (LECs), whereas closed junctions in collecting LECs prevent lymph leakage. LEC junctions are known to dynamically remodel in development and disease, but how lymphatic permeability is regulated remains poorly understood.METHODS: We used various genetically engineered mouse models in combination with cellular, biochemical, and molecular biology approaches to elucidate the signaling pathways regulating junction morphology and function in lymphatic capillaries.
    RESULTS: By studying the permeability of intestinal lacteal capillaries to lipoprotein particles known as chylomicrons, we show that ROCK (Rho-associated kinase)-dependent cytoskeletal contractility is a fundamental mechanism of LEC permeability regulation. We show that chylomicron-derived lipids trigger neonatal lacteal junction opening via ROCK-dependent contraction of junction-anchored stress fibers. LEC-specific ROCK deletion abolished junction opening and plasma lipid uptake. Chylomicrons additionally inhibited VEGF (vascular endothelial growth factor)-A signaling. We show that VEGF-A antagonizes LEC junction opening via VEGFR (VEGF receptor) 2 and VEGFR3-dependent PI3K/AKT activation of the small GTPase RAC1, thereby restricting RhoA/ROCK-mediated cytoskeleton contraction.
    CONCLUSIONS: Our results reveal that antagonistic inputs into ROCK-dependent cytoskeleton contractions regulate the interconversion of lymphatic junctions in the intestine and in other tissues, providing a tunable mechanism to control the lymphatic barrier.
    Keywords:  chylomicrons; endothelial cell; lipid; permeability; vascular endothelial growth factor
    DOI:  https://doi.org/10.1161/CIRCRESAHA.123.322607
This page is being maintained by Thomas Krichel. It was last updated on 2023‒07‒23 at 9:47.