bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–06–19
twenty-two papers selected by
Ralitsa Radostinova Madsen, University College London
  1. PI3King the Environment for Growth: PI3K Activation Drives Transcriptome Changes That Support Oncogenic Growth.
  2. The onset of PI3K-related vascular malformations occurs during angiogenesis and is prevented by the AKT inhibitor miransertib.
  3. An isogenic cell line panel for sequence-based screening of targeted anticancer drugs.
  4. Development of a physiological insulin resistance model in human stem cell-derived adipocytes.
  5. Mass spectrometry-based draft of the mouse proteome.
  6. Optogenetic actuator - ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics.
  7. FOXA2 suppresses endometrial carcinogenesis and epithelial-mesenchymal transition by regulating enhancer activity.
  8. Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer.
  9. IFN-I signaling in cancer: the connection with dysregulated Insulin/IGF axis.
  10. PI3K/Akt pathway mediates the positive inotropic effects of insulin in Langendorff-perfused rat hearts.
  11. Unbiased spatial proteomics with single-cell resolution in tissues.
  12. S.U.G.A.R: A Case to Outline Tactics for the Prevention of Alpelisib-Induced Hyperglycemia.
  13. Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing.
  14. Spatial profiling of early primate gastrulation in utero.
  15. Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells.
  16. Comprehensive Analysis Identifies PI3K/Akt Pathway Alternations as an Immune-Related Prognostic Biomarker in Colon Adenocarcinoma Patients Receiving Immune Checkpoint Inhibitor Treatment.
  17. Reading oscillatory instructions: How cells achieve time-dependent responses to oscillating transcription factors.
  18. Signatures of copy number alterations in human cancer.
  19. Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR.
  20. Metabolic routing maintains the unique fatty acid composition of phosphoinositides.
  21. A hexa-species transcriptome atlas of mammalian embryogenesis delineates metabolic regulation across three different implantation modes.
  22. Immortalised Cas9-expressing Cell lines for Gene interrogation.
  1. Cancer Res. 2022 Jun 15. 82(12): 2216-2218
    PI3King the Environment for Growth: PI3K Activation Drives Transcriptome Changes That Support Oncogenic Growth.
    Sally E Claridge, Benjamin D Hopkins.
      PI3K signaling plays an integral role in cells, coordinating the necessary alterations in cellular metabolism and programs to support survival and proliferation. In the first genome-wide analysis of alternative splicing in PIK3CA-mutant breast cancer, Ladewig and colleagues show that activating mutations in PIK3CA alter the use of known exons and splice junctions, leading to changes in gene expression and transcription factor activity that promote an oncogenic phenotype. Their work reveals a novel mechanism underlying the functional impact of PI3K signal activation in the context of breast cancer, where PIK3CA mutations are common and PI3K inhibitors are part of the standard of care. Their studies uncover a feedforward mechanism by which PI3K signaling enables a shift in the spectrum of translated splice variants as another method through which the PI3K pathway has evolved to regulate its own activity, thereby modifying the cellular response to upstream activation based on the signaling that has come before. These findings have profound implications for understanding the evolution of the PI3K pathway and the rewiring of cells in response to prolonged or repeated signal activation. See related article by Ladewig et al., p. 2269.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-1466
  2. EMBO Mol Med. 2022 Jun 13. e15619
    The onset of PI3K-related vascular malformations occurs during angiogenesis and is prevented by the AKT inhibitor miransertib.
    Piotr Kobialka, Helena Sabata, Odena Vilalta, Leonor Gouveia, Ana Angulo-Urarte, Laia Muixí, Jasmina Zanoncello, Oscar Muñoz-Aznar, Nagore G Olaciregui, Lucia Fanlo, Anna Esteve-Codina, Cinzia Lavarino, Biola M Javierre, Veronica Celis, Carlota Rovira, Susana López-Fernández, Eulàlia Baselga, Jaume Mora, Sandra D Castillo, Mariona Graupera.
      Low-flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3caH1047R activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K-driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K-driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human low-flow vascular malformations.
    Keywords:  AKT; PI3K; angiogenesis; endothelial cell; vascular malformations
    DOI:  https://doi.org/10.15252/emmm.202115619
  3. iScience. 2022 Jun 17. 25(6): 104437
    An isogenic cell line panel for sequence-based screening of targeted anticancer drugs.
    Ashley L Cook, Nicolas Wyhs, Surojit Sur, Blair Ptak, Maria Popoli, Laura Dobbyn, Tasos Papadopoulos, Chetan Bettegowda, Nickolas Papadopoulos, Bert Vogelstein, Shibin Zhou, Kenneth W Kinzler.
      We describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.
    Keywords:  Biochemical analysis; Biochemistry; Cancer
    DOI:  https://doi.org/10.1016/j.isci.2022.104437
  4. Sci Adv. 2022 Jun 17. 8(24): eabn7298
    Development of a physiological insulin resistance model in human stem cell-derived adipocytes.
    Max Friesen, Andrew S Khalil, M Inmaculada Barrasa, Jacob F Jeppesen, David J Mooney, Rudolf Jaenisch.
      Adipocytes are key regulators of human metabolism, and their dysfunction in insulin signaling is central to metabolic diseases including type II diabetes mellitus (T2D). However, the progression of insulin resistance into T2D is still poorly understood. This limited understanding is due, in part, to the dearth of suitable models of insulin signaling in human adipocytes. Traditionally, adipocyte models fail to recapitulate in vivo insulin signaling, possibly due to exposure to supraphysiological nutrient and hormone conditions. We developed a protocol for human pluripotent stem cell-derived adipocytes that uses physiological nutrient conditions to produce a potent insulin response comparable to in vivo adipocytes. After systematic optimization, this protocol allows robust insulin-stimulated glucose uptake and transcriptional insulin response. Furthermore, exposure of sensitized adipocytes to physiological hyperinsulinemia dampens insulin-stimulated glucose uptake and dysregulates insulin-responsive transcription. Overall, our methodology provides a novel platform for the mechanistic study of insulin signaling and resistance using human pluripotent stem cell-derived adipocytes.
    DOI:  https://doi.org/10.1126/sciadv.abn7298
  5. Nat Methods. 2022 Jun 16.
    Mass spectrometry-based draft of the mouse proteome.
    Piero Giansanti, Patroklos Samaras, Yangyang Bian, Chen Meng, Andrea Coluccio, Martin Frejno, Hannah Jakubowsky, Sophie Dobiasch, Rashmi R Hazarika, Julia Rechenberger, Julia Calzada-Wack, Johannes Krumm, Sebastian Mueller, Chien-Yun Lee, Nicole Wimberger, Ludwig Lautenbacher, Zonera Hassan, Yun-Chien Chang, Chiara Falcomatà, Florian P Bayer, Stefanie Bärthel, Tobias Schmidt, Roland Rad, Stephanie E Combs, Matthew The, Frank Johannes, Dieter Saur, Martin Hrabe de Angelis, Mathias Wilhelm, Günter Schneider, Bernhard Kuster.
      The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.
    DOI:  https://doi.org/10.1038/s41592-022-01526-y
  6. Mol Syst Biol. 2022 Jun;18(6): e10670
    Optogenetic actuator - ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics.
    Coralie Dessauges, Jan Mikelson, Maciej Dobrzyński, Marc-Antoine Jacques, Agne Frismantiene, Paolo Armando Gagliardi, Mustafa Khammash, Olivier Pertz.
      Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.
    Keywords:  ERK dynamics; MAPK network; optogenetics; signaling robustness; single-cell biology
    DOI:  https://doi.org/10.15252/msb.202110670
  7. J Clin Invest. 2022 Jun 15. pii: e157574. [Epub ahead of print]132(12):
    FOXA2 suppresses endometrial carcinogenesis and epithelial-mesenchymal transition by regulating enhancer activity.
    Subhransu S Sahoo, Susmita G Ramanand, Yunpeng Gao, Ahmed Abbas, Ashwani Kumar, Ileana C Cuevas, Hao-Dong Li, Mitzi Aguilar, Chao Xing, Ram S Mani, Diego H Castrillon.
      FOXA2 encodes a transcription factor mutated in 10% of endometrial cancers (ECs), with a higher mutation rate in aggressive variants. FOXA2 has essential roles in embryonic and uterine development. However, FOXA2's role in EC is incompletely understood. Functional investigations using human and mouse EC cell lines revealed that FOXA2 controls endometrial epithelial gene expression programs regulating cell proliferation, adhesion, and endometrial-epithelial transition. In live animals, conditional inactivation of Foxa2 or Pten alone in endometrial epithelium did not result in ECs, but simultaneous inactivation of both genes resulted in lethal ECs with complete penetrance, establishing potent synergism between Foxa2 and PI3K signaling. Studies in tumor-derived cell lines and organoids highlighted additional invasion and cell growth phenotypes associated with malignant transformation and identified key mediators, including Myc and Cdh1. Transcriptome and cistrome analyses revealed that FOXA2 broadly controls gene expression programs through modification of enhancer activity in addition to regulating specific target genes, rationalizing its tumor suppressor functions. By integrating results from our cell lines, organoids, animal models, and patient data, our findings demonstrated that FOXA2 is an endometrial tumor suppressor associated with aggressive disease and with shared commonalities among its roles in endometrial function and carcinogenesis.
    Keywords:  Cancer; Molecular pathology; Oncology; Tumor suppressors
    DOI:  https://doi.org/10.1172/JCI157574
  8. STAR Protoc. 2022 Jun 17. 3(2): 101438
    Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer.
    Fabiana Lüönd, Natascha Santacroce, Christian Beisel, Laurent Guérard, Thomas R Bürglin, Gerhard Christofori, Nami Sugiyama.
      The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021).
    Keywords:  Cancer; Cell Biology; Cell Differentiation; Flow Cytometry/Mass Cytometry; Microscopy; Molecular Biology; RNAseq; Single Cell
    DOI:  https://doi.org/10.1016/j.xpro.2022.101438
  9. Trends Endocrinol Metab. 2022 Jun 09. pii: S1043-2760(22)00082-0. [Epub ahead of print]
    IFN-I signaling in cancer: the connection with dysregulated Insulin/IGF axis.
    Veronica Vella, Ernestina Marianna De Francesco, Eduardo Bonavita, Rosamaria Lappano, Antonino Belfiore.
      Type I interferons (IFN-Is) are prototypical inflammatory cytokines produced in response to stress. IFN-Is have a critical role in antitumor immunity by driving the activation of leukocytes and favoring the elimination of malignant cells. However, IFN-I signaling in cancer, specifically in the tumor microenvironment (TME), can have opposing roles. Sustained IFN-I stimulation can promote immune exhaustion or enable tumor cell-intrinsic malignant features. Herein, we discuss the potential impact of the insulin/insulin-like growth factor system (I/IGFs) and of metabolic disorders in aberrant IFN-I signaling in cancer. We consider the possibility that targeting I/IGFs, especially in patients with cancer affected by metabolic disorders, contributes to an effective strategy to inhibit deleterious IFN-I signaling, thereby restoring sensitivity to various cancer therapies, including immunotherapy.
    Keywords:  IGF axis; insulin receptor isoform A; insulin receptor isoforms; interferon signaling
    DOI:  https://doi.org/10.1016/j.tem.2022.04.009
  10. Sci Rep. 2022 Jun 13. 12(1): 9793
    PI3K/Akt pathway mediates the positive inotropic effects of insulin in Langendorff-perfused rat hearts.
    Yosuke Nakadate, Akiko Kawakami, Hiroaki Sato, Tamaki Sato, Takeshi Oguchi, Keisuke Omiya, Toru Matsuoka, Thomas Schricker, Takashi Matsukawa.
      Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt) activation remains unclear. We hypothesized that the positive inotropic effects of insulin are dose-dependent and mediated via the PI3K/Akt pathway in isolated normal rat hearts. The Institutional Animal Investigation Committee approved the use of hearts excised from rats under pentobarbital anesthesia. The hearts were perfused at a constant pressure using the Langendorff technique. After stabilization (baseline), the hearts were randomly divided into the following four insulin (Ins) groups: 1) Ins0 (0 IU/L), 2) Ins0.5 (0.5 IU/L), 3) Ins5 (5 IU/L), and 4) Ins50 (50 IU/L) (n = 8 in each group). To clarify the role of the PI3K/Akt pathway in insulin-dependent inotropic effects, we also treated the insulin groups with the PI3K inhibitor wortmannin (InsW): 5) InsW0 (0 IU/L), 6) InsW0.5 (0.5 IU/L), 7) InsW5 (5 IU/L), and 8) InsW50 (50 IU/L). Hearts were perfused with Krebs-Henseleit buffer solution with or without wortmannin for 10 min, followed by 20 min perfusion with the solution containing each concentration of insulin. The data were recorded as the maximum left ventricular derivative of pressure development (LV dP/dt max). Myocardial p-Akt levels were measured at 3 min, 5 min, and at the end of the perfusion. In the Ins groups, LV dP/dt max in Ins5 and Ins50 increased by 14% and 48%, respectively, 3 min after insulin perfusion compared with the baseline. Tachyphylaxis was observed after 10 min in the Ins5 and Ins50 treatment groups. Wortmannin partially inhibited the positive inotropic effect of insulin; although insulin enhanced p-Akt levels at all time points compared with the control group, this increase was suppressed in the presence of wortmannin. The positive inotropic effect of insulin is dose-dependent and consistent with Akt activation. This effect mediated by high doses of insulin on cardiac tissue was temporary and caused tachyphylaxis, potentially triggered by Akt overactivation, which leads beta 1 deactivation.
    DOI:  https://doi.org/10.1038/s41598-022-14092-2
  11. Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00489-0. [Epub ahead of print]82(12): 2335-2349
    Unbiased spatial proteomics with single-cell resolution in tissues.
    Andreas Mund, Andreas-David Brunner, Matthias Mann.
      Mass spectrometry (MS)-based proteomics has become a powerful technology to quantify the entire complement of proteins in cells or tissues. Here, we review challenges and recent advances in the LC-MS-based analysis of minute protein amounts, down to the level of single cells. Application of this technology revealed that single-cell transcriptomes are dominated by stochastic noise due to the very low number of transcripts per cell, whereas the single-cell proteome appears to be complete. The spatial organization of cells in tissues can be studied by emerging technologies, including multiplexed imaging and spatial transcriptomics, which can now be combined with ultra-sensitive proteomics. Combined with high-content imaging, artificial intelligence and single-cell laser microdissection, MS-based proteomics provides an unbiased molecular readout close to the functional level. Potential applications range from basic biological questions to precision medicine.
    DOI:  https://doi.org/10.1016/j.molcel.2022.05.022
  12. J Investig Med High Impact Case Rep. 2022 Jan-Dec;10:10 23247096221105249
    S.U.G.A.R: A Case to Outline Tactics for the Prevention of Alpelisib-Induced Hyperglycemia.
    Katharine Thomas, Monique Germain, Michelle M Loch.
      Postmenopausal patients with metastatic breast cancer (mBC) may live years with their disease on therapies with minimal toxicities but they will eventually progress on first-line therapy. For those eligible for second-line therapy, PIK3CA mutation testing is recommended in estrogen receptor-positive, her2-negative disease. If present, alpelisib, a PI3K inhibitor, has been shown to improve progression-free survival. Hyperglycemia is a common side effect of alpelisib. We describe a case of diabetic ketoacidosis (DKA) necessitating treatment in the intensive care unit (ICU) in a woman with type 2 diabetes mellitus (T2DM) started on alpelisib. A 76-year-old female with diet-controlled T2DM and mBC was placed on second-line treatment with alpelisib after progression on first-line therapy. After more than 2 weeks of treatment, the patient presented to the emergency department with nausea and vomiting. Lab results showed DKA and she was admitted to the ICU for further management. This case highlights the need for a multidisciplinary approach to caring for patients who are started on a PI3K inhibitor. We propose 5 guidelines to prevent hyperglycemia in those started on apelisib: (1) strict criteria for initiating alpelisib, (2) understand the steps needed to prevent hyperglycemia, (3) get help from a multidisciplinary team, (4) act immediately when hyperglycemia is noted, and (5) record blood glucose values. By implementing these steps, we hope to prevent critical hyperglycemic episodes in vulnerable patients on alpelisib.
    Keywords:  breast cancer; hematology/oncology; pharmacology
    DOI:  https://doi.org/10.1177/23247096221105249
  13. Proteomics. 2022 Jun 17. e2100245
    Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing.
    Claire Koenig, Ana Martinez-Val, Giulia Franciosa, Jesper V Olsen.
      In large-scale quantitative mass spectrometry (MS)-based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high-pH reversed-phase peptide chromatographic fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affect sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT-based fractionation strategies against single-shot label-free approach with spectral library-free data independent acquisition (LFQ-DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT-fractionation approaches in a phosphoproteomics workflow, we compared two different high-pH reverse-phase fractionation strategies, microflow (MF) and stage-tip fractionation (STF), while scaling the peptide input amount down from 12.5 μg to 1 μg per sample. Our results indicate that, for input amounts higher than 5 μg per sample, TMT labeling, followed by microflow fractionation and phospho-enrichment (MF), achieves the deepest phosphoproteome coverage, even compared to single shot direct-DIA analysis. Conversely, stage-tip fractionation of enriched phosphopeptides (STF) is optimal for lower amounts, below 5 μg/peptide per sample. As a result, we provide a decision tree to help phosphoproteomics users to choose the best workflow as a function of on sample amount. This article is protected by copyright. All rights reserved.
    Keywords:  high-pH fractionation; isobaric labeling; phosphoproteomics; scale-down; tandem mass tags
    DOI:  https://doi.org/10.1002/pmic.202100245
  14. Nature. 2022 Jun 16.
    Spatial profiling of early primate gastrulation in utero.
    Sophie Bergmann, Christopher A Penfold, Erin Slatery, Dylan Siriwardena, Charis Drummer, Stephen Clark, Stanley E Strawbridge, Keiko Kishimoto, Alice Vickers, Mukul Tewary, Timo N Kohler, Florian Hollfelder, Wolf Reik, Erika Sasaki, Rüdiger Behr, Thorsten E Boroviak.
      Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1-3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4-6 and to advance stem-cell-based regenerative approaches7. Here, we illuminate early gastrulation of marmoset embryos in utero by spatial transcriptomics and stem cell-based embryo models. Gaussian process regression-based 3D-transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates primitive streak formation in the embryonic disc and is counteracted by SFRP1/2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1/2/3 in response to BMP-signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit highest similarity to the anterior embryonic disc, while naïve PSCs resemble the preimplantation epiblast. Our 3D-transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
    DOI:  https://doi.org/10.1038/s41586-022-04953-1
  15. Mol Ther Nucleic Acids. 2022 Jun 14. 28 877-891
    Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells.
    Taekyu Ha, Michael DiPrima, Vishal Koparde, Parthav Jailwala, Hidetaka Ohnuki, Jing-Xin Feng, Murali Palangat, Daniel Larson, Giovanna Tosato.
      Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.
    Keywords:  MT: Oligonucleotides; Therapies and Applications; antisense reads; colorectal cancer; ephrinB2; gene therapy; integrated stress response; lentiviral vector; shRNA; vector integration
    DOI:  https://doi.org/10.1016/j.omtn.2022.05.029
  16. J Immunol Res. 2022 ;2022 8179799
    Comprehensive Analysis Identifies PI3K/Akt Pathway Alternations as an Immune-Related Prognostic Biomarker in Colon Adenocarcinoma Patients Receiving Immune Checkpoint Inhibitor Treatment.
    Anqi Lin, Tianqi Gu, Xigang Hu, Jian Zhang, Peng Luo.
       Introduction: In recent years, immune checkpoint inhibitors (ICIs) have attracted widespread attention and made breakthroughs in progress towards the treatment of various cancers. However, ICI therapy is selective, and its effects on many patients are not ideal. It is therefore critical to identify prognostic biomarkers of response to ICI therapy. The PI3K/Akt pathway plays important roles in tumor formation and metastasis. However, there are no published reports clarifying the relationship between PI3K/Akt pathway mutations and prognosis for colon adenocarcinoma (COAD) patients receiving immunotherapy.
    Methods: We collected data from a COAD cohort from The Cancer Genome Atlas (TCGA) database, including whole-exome sequencing (WES) data, RNA-seq data, and clinical data. We also collected data, including clinical prognosis and targeted sequencing data, from a cohort of COAD patients receiving immunotherapy. We collected 50 COAD patients (Local-COAD) from the Zhujiang Hospital of Southern Medical University and performed targeted sequencing. We analyzed the effects of PI3K/Akt pathway mutations on the patients' clinical prognosis, immunogenicity, and immune microenvironments. Gene set enrichment analysis (GSEA) was used to analyze the significantly upregulated and downregulated signaling pathways. We used these results to hypothesize potential mechanisms by which PI3K/Akt mutations could affect the prognosis of COAD patients.
    Results: Univariate and multivariate Cox analyses and Kaplan-Meier (KM) survival curves showed that patients with PI3K-Akt mutations had better overall survival (OS) than those without PI3K-Akt mutations. Genes with significant mutation rates in the two cohorts were screened by panoramic view. CIBERSORT was used to analyze changes in 22 types of immune cells to identify immune activated cells. Similarly, patients in the PI3K/Akt-mutated type (PI3K/Akt-MT) group had significantly increased immunogenicity, including increases in tumor mutation burden (TMB), neoantigen load (NAL), and MANTIS score. Using GSEA, we identified upregulated pathways related to immune response.
    Conclusion: PI3K/Akt pathway mutation status can be used as an independent predictor of response to ICI treatment in COAD patients. PI3K/Akt mutations are correlated with improved OS, higher immunogenicity, greater immune response scores, and increases in activated immune cells.
    DOI:  https://doi.org/10.1155/2022/8179799
  17. Curr Opin Cell Biol. 2022 Jun 08. pii: S0955-0674(22)00052-7. [Epub ahead of print]77 102099
    Reading oscillatory instructions: How cells achieve time-dependent responses to oscillating transcription factors.
    Veena Venkatachalam, Ashwini Jambhekar, Galit Lahav.
      
    DOI:  https://doi.org/10.1016/j.ceb.2022.102099
  18. Nature. 2022 Jun 15.
    Signatures of copy number alterations in human cancer.
    Christopher D Steele, Ammal Abbasi, S M Ashiqul Islam, Amy L Bowes, Azhar Khandekar, Kerstin Haase, Shadi Hames-Fathi, Dolapo Ajayi, Annelien Verfaillie, Pawan Dhami, Alex McLatchie, Matt Lechner, Nicholas Light, Adam Shlien, David Malkin, Andrew Feber, Paula Proszek, Tom Lesluyes, Fredrik Mertens, Adrienne M Flanagan, Maxime Tarabichi, Peter Van Loo, Ludmil B Alexandrov, Nischalan Pillay.
      Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage-fusion-bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3-5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.
    DOI:  https://doi.org/10.1038/s41586-022-04738-6
  19. Cell Rep. 2022 Jun 14. pii: S2211-1247(22)00732-X. [Epub ahead of print]39(11): 110950
    Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR.
    Mireia Perez Verdaguer, Tian Zhang, Sachin Surve, Joao A Paulo, Callen Wallace, Simon C Watkins, Steven P Gygi, Alexander Sorkin.
      Ligand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.
    Keywords:  CP: Molecular biology; EGF receptor; endocytosis; proximity labeling; signaling
    DOI:  https://doi.org/10.1016/j.celrep.2022.110950
  20. EMBO Rep. 2022 Jun 16. e54532
    Metabolic routing maintains the unique fatty acid composition of phosphoinositides.
    Yeun Ju Kim, Nivedita Sengupta, Mira Sohn, Amrita Mandal, Joshua G Pemberton, Uimook Choi, Tamas Balla.
      Phosphoinositide lipids (PPIn) are enriched in stearic- and arachidonic acids (38:4) but how this enrichment is established and maintained during phospholipase C (PLC) activation is unknown. Here we show that the metabolic fate of newly synthesized phosphatidic acid (PA), the lipid precursor of phosphatidylinositol (PI), is influenced by the fatty acyl-CoA used with preferential routing of the arachidonoyl-enriched species toward PI synthesis. Furthermore, during agonist stimulation the unsaturated forms of PI(4,5P)2 are replenished significantly faster than the more saturated ones, suggesting a favored recycling of the unsaturated forms of the PLC-generated hydrolytic products. Cytidine diphosphate diacylglycerol synthase 2 (CDS2) but not CDS1 was found to contribute to increased PI resynthesis during PLC activation. Lastly, while the lipid transfer protein, Nir2 is found to contribute to rapid PPIn resynthesis during PLC activation, the faster re-synthesis of the 38:4 species does not depend on Nir2. Therefore, the fatty acid side-chain composition of the lipid precursors used for PI synthesis is an important determinant of their metabolic fates, which also contributes to the maintenance of the unique fatty acid profile of PPIn lipids.
    Keywords:  diacylglycerol; lipid transfer proteins; phosphatidic acid; phosphatidylinositol; phospholipase C
    DOI:  https://doi.org/10.15252/embr.202154532
  21. Nat Commun. 2022 Jun 16. 13(1): 3407
    A hexa-species transcriptome atlas of mammalian embryogenesis delineates metabolic regulation across three different implantation modes.
    Anna Malkowska, Christopher Penfold, Sophie Bergmann, Thorsten E Boroviak.
      Mammalian embryogenesis relies on glycolysis and oxidative phosphorylation to balance the generation of biomass with energy production. However, the dynamics of metabolic regulation in the postimplantation embryo in vivo have remained elusive due to the inaccessibility of the implanted conceptus for biochemical studies. To address this issue, we compiled single-cell embryo profiling data in six mammalian species and determined their metabolic dynamics through glycolysis and oxidative phosphorylation associated gene expression. Strikingly, we identify a conserved switch from bivalent respiration in the late blastocyst towards a glycolytic metabolism in early gastrulation stages across species, which is independent of embryo implantation. Extraembryonic lineages followed the dynamics of the embryonic lineage, except visceral endoderm. Finally, we demonstrate that in vitro primate embryo culture substantially impacts metabolic gene regulation by comparison to in vivo samples. Our work reveals a conserved metabolic programme despite different implantation modes and highlights the need to optimise postimplantation embryo culture protocols.
    DOI:  https://doi.org/10.1038/s41467-022-30194-x
  22. Methods Mol Biol. 2022 ;2495 91-97
    Immortalised Cas9-expressing Cell lines for Gene interrogation.
    Luis F Malaver-Ortega, Joseph Rosenbluh.
      The ability of modifying the genome of multiple species, precisely and without or minimal off-targeted effects, have opened numerous opportunities for the biotechnology industry. In this chapter, we describe an easy to establish, robust, and practical pipeline that can be used to generate immortalized cell lines, from different tissues, to capture cell linage context and validate the tools required for genome editing and genetic modification. This pipeline serves as a reference for similar approaches for gene interrogation in other species.
    Keywords:  CRISPR/Cas9; Cas9 assay; Cell immortalization; Genome editing
    DOI:  https://doi.org/10.1007/978-1-0716-2301-5_5
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