bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–03–17
sixteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. AlphaPept: a modern and open framework for MS-based proteomics.
  2. High-throughput evaluation of genetic variants with prime editing sensor libraries.
  3. PTEN loss in glioma cell lines leads to increased extracellular vesicles biogenesis and PD-L1 cargo in a PI3K-dependent manner.
  4. Continuous evolution of compact protein degradation tags regulated by selective molecular glues.
  5. Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.
  6. Accelerating Single-Cell Sequencing Data Analysis with SciDAP: A User-Friendly Approach.
  7. Charting cellular differentiation trajectories with Ricci flow.
  8. A temperature-inducible protein module for control of mammalian cell fate.
  9. Lymphatic malformations: mechanistic insights and evolving therapeutic frontiers.
  10. Distinct early role of PTEN regulation during HCMV infection of monocytes.
  11. The Integrated Stress Response in metabolic adaptation.
  12. Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.
  13. NCI Cancer Research Data Commons: Cloud-based Analytical Resources.
  14. NCI Cancer Research Data Commons: Resources to Share Key Cancer Data.
  15. Modular vector assembly enables rapid assessment of emerging CRISPR technologies.
  16. A neural network-based model framework for cell-fate decisions and development.
  1. Nat Commun. 2024 Mar 09. 15(1): 2168
    AlphaPept: a modern and open framework for MS-based proteomics.
    Maximilian T Strauss, Isabell Bludau, Wen-Feng Zeng, Eugenia Voytik, Constantin Ammar, Julia P Schessner, Rajesh Ilango, Michelle Gill, Florian Meier, Sander Willems, Matthias Mann.
      In common with other omics technologies, mass spectrometry (MS)-based proteomics produces ever-increasing amounts of raw data, making efficient analysis a principal challenge. A plethora of different computational tools can process the MS data to derive peptide and protein identification and quantification. However, during the last years there has been dramatic progress in computer science, including collaboration tools that have transformed research and industry. To leverage these advances, we develop AlphaPept, a Python-based open-source framework for efficient processing of large high-resolution MS data sets. Numba for just-in-time compilation on CPU and GPU achieves hundred-fold speed improvements. AlphaPept uses the Python scientific stack of highly optimized packages, reducing the code base to domain-specific tasks while accessing the latest advances. We provide an easy on-ramp for community contributions through the concept of literate programming, implemented in Jupyter Notebooks. Large datasets can rapidly be processed as shown by the analysis of hundreds of proteomes in minutes per file, many-fold faster than acquisition. AlphaPept can be used to build automated processing pipelines with web-serving functionality and compatibility with downstream analysis tools. It provides easy access via one-click installation, a modular Python library for advanced users, and via an open GitHub repository for developers.
    DOI:  https://doi.org/10.1038/s41467-024-46485-4
  2. Nat Biotechnol. 2024 Mar 12.
    High-throughput evaluation of genetic variants with prime editing sensor libraries.
    Samuel I Gould, Alexandra N Wuest, Kexin Dong, Grace A Johnson, Alvin Hsu, Varun K Narendra, Ondine Atwa, Stuart S Levine, David R Liu, Francisco J Sánchez Rivera.
      Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.
    DOI:  https://doi.org/10.1038/s41587-024-02172-9
  3. bioRxiv. 2024 Mar 02. pii: 2023.07.26.550575. [Epub ahead of print]
    PTEN loss in glioma cell lines leads to increased extracellular vesicles biogenesis and PD-L1 cargo in a PI3K-dependent manner.
    Julio C Sanchez, Timothy M Pierpont, Dariana Argueta-Zamora, Kristin Wilson, Avery August, Richard A Cerione.
      Phosphatase and Tensin Homologue (PTEN) is one of the most frequently lost tumor suppressors in cancer and the predominant negative regulator of the PI3K/AKT signaling axis. A growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1), an altered tumor derived secretome that drives an immunosuppressive tumor immune microenvironment (TIME), and resistance to certain immunotherapies. Given their roles in immunosuppression and tumor growth, we examined whether the loss of PTEN would impact the biogenesis, cargo, and function of extracellular vesicles (EVs) in the context of the anti-tumor associated cytokine interferon-γ (IFN-γ). Through genetic and pharmacological approaches, we show that PD-L1 expression is regulated by JAK/STAT signaling, not PI3K signaling. Instead, we observe that PTEN loss positively upregulates cell surface levels of PD-L1 and enhances the biogenesis of EVs enriched with PD-L1 in a PI3K-dependent manner. We demonstrate that because of these changes, EVs derived from glioma cells lacking PTEN have a greater ability to suppress T cell receptor (TCR) signaling. Taken together, these findings provide important new insights into how the loss of PTEN can contribute to an immunosuppressive TIME, facilitate immune evasion, and highlight a novel role for PI3K signaling in the regulation of EV biogenesis and the cargo they contain.
    DOI:  https://doi.org/10.1101/2023.07.26.550575
  4. Science. 2024 Mar 15. 383(6688): eadk4422
    Continuous evolution of compact protein degradation tags regulated by selective molecular glues.
    Jaron A M Mercer, Stephan J DeCarlo, Shourya S Roy Burman, Vedagopuram Sreekanth, Andrew T Nelson, Moritz Hunkeler, Peter J Chen, Katherine A Donovan, Praveen Kokkonda, Praveen K Tiwari, Veronika M Shoba, Arghya Deb, Amit Choudhary, Eric S Fischer, David R Liu.
      Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.
    DOI:  https://doi.org/10.1126/science.adk4422
  5. In Vitro Cell Dev Biol Anim. 2024 Mar 12.
    Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.
    Japanese Working Group for Consideration of Good Cell Culture Practice
      Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.
    Keywords:  Cell culture; Diversity; Good Cell Culture Practice; Pluripotent stem cell
    DOI:  https://doi.org/10.1007/s11626-024-00863-w
  6. bioRxiv. 2024 Feb 29. pii: 2024.02.28.582604. [Epub ahead of print]
    Accelerating Single-Cell Sequencing Data Analysis with SciDAP: A User-Friendly Approach.
    Michael Kotliar, Andrey Kartashov, Artem Barski.
      Single-cell (sc) RNA, ATAC and Multiome sequencing became powerful tools for uncovering biological and disease mechanisms. Unfortunately, manual analysis of sc data presents multiple challenges due to large data volumes and complexity of configuration parameters. This complexity, as well as not being able to reproduce a computational environment, affects the reproducibility of analysis results. The Scientific Data Analysis Platform ( https://SciDAP.com ) allows biologists without computational expertise to analyze sequencing-based data using portable and reproducible pipelines written in Common Workflow Language (CWL). Our suite of computational pipelines addresses the most common needs in scRNA-Seq, scATAC-Seq and scMultiome data analysis. When executed on SciDAP, it offers a user-friendly alternative to manual data processing, eliminating the need for coding expertise. In this protocol, we describe the use of SciDAP to analyze scMultiome data. Similar approaches can be used for analysis of scRNA-Seq, scATAC-Seq and scVDJ-Seq datasets.
    DOI:  https://doi.org/10.1101/2024.02.28.582604
  7. Nat Commun. 2024 Mar 13. 15(1): 2258
    Charting cellular differentiation trajectories with Ricci flow.
    Anthony Baptista, Ben D MacArthur, Christopher R S Banerji.
      Complex biological processes, such as cellular differentiation, require intricate rewiring of intra-cellular signalling networks. Previous characterisations revealed a raised network entropy underlies less differentiated and malignant cell states. A connection between entropy and Ricci curvature led to applications of discrete curvatures to biological networks. However, predicting dynamic biological network rewiring remains an open problem. Here we apply Ricci curvature and Ricci flow to biological network rewiring. By investigating the relationship between network entropy and Forman-Ricci curvature, theoretically and empirically on single-cell RNA-sequencing data, we demonstrate that the two measures do not always positively correlate, as previously suggested, and provide complementary rather than interchangeable information. We next employ Ricci flow to derive network rewiring trajectories from stem cells to differentiated cells, accurately predicting true intermediate time points in gene expression time courses. In summary, we present a differential geometry toolkit for understanding dynamic network rewiring during cellular differentiation and cancer.
    DOI:  https://doi.org/10.1038/s41467-024-45889-6
  8. bioRxiv. 2024 Feb 19. pii: 2024.02.19.581019. [Epub ahead of print]
    A temperature-inducible protein module for control of mammalian cell fate.
    William Benman, Zikang Dennis Huang, Pavan Iyengar, Delaney Wilde, Thomas Mumford, Lukasz Bugaj.
      Inducible protein switches are used throughout the biosciences to allow on-demand control of proteins in response to chemical or optical inputs. However, these inducers either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization through temperature), exists as a monomer in the cytoplasm at elevated temperatures but both oligomerizes and translocates to the plasma membrane when temperature is lowered. Using custom devices for rapid and high-throughput temperature control during live-cell microscopy, we find that the original Melt variant fully switches states between 28-32C, and state changes can be observed within minutes of temperature changes. Melt was highly modular, permitting thermal control over diverse intracellular processes including signaling, proteolysis, and nuclear shuttling through straightforward end-to-end fusions with no further engineering. Melt was also highly tunable, giving rise to a library of Melt variants with switch point temperatures ranging from 30-40C. The variants with higher switch points allowed control of molecular circuits between 37C-41C, a well-tolerated range for mammalian cells. Finally, Melt could thermally regulate important cell decisions over this range, including cytoskeletal rearrangement and apoptosis. Thus Melt represents a versatile thermogenetic module that provides straightforward, temperature-based, real-time control of mammalian cells with broad potential for biotechnology and biomedicine.
    DOI:  https://doi.org/10.1101/2024.02.19.581019
  9. J Clin Invest. 2024 Mar 15. pii: e172844. [Epub ahead of print]134(6):
    Lymphatic malformations: mechanistic insights and evolving therapeutic frontiers.
    Milena Petkova, Ingvar Ferby, Taija Mäkinen.
      The lymphatic vascular system is gaining recognition for its multifaceted role and broad pathological significance. Once perceived as a mere conduit for interstitial fluid and immune cell transport, recent research has unveiled its active involvement in critical physiological processes and common diseases, including inflammation, autoimmune diseases, and atherosclerosis. Consequently, abnormal development or functionality of lymphatic vessels can result in serious health complications. Here, we discuss lymphatic malformations (LMs), which are localized lesions that manifest as fluid-filled cysts or extensive infiltrative lymphatic vessel overgrowth, often associated with debilitating, even life-threatening, consequences. Genetic causes of LMs have been uncovered, and several promising drug-based therapies are currently under investigation and will be discussed.
    DOI:  https://doi.org/10.1172/JCI172844
  10. Proc Natl Acad Sci U S A. 2024 Mar 19. 121(12): e2312290121
    Distinct early role of PTEN regulation during HCMV infection of monocytes.
    Liudmila S Chesnokova, Bailey S Mosher, Heather L Fulkerson, Hyung W Nam, Akhalesh K Shakya, Andrew D Yurochko.
      Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to β1/β3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.
    Keywords:  HCMV; cellular trafficking; monocytes; phosphoproteomics; signaling
    DOI:  https://doi.org/10.1073/pnas.2312290121
  11. J Biol Chem. 2024 Mar 08. pii: S0021-9258(24)01646-6. [Epub ahead of print] 107151
    The Integrated Stress Response in metabolic adaptation.
    Hyung Don Ryoo.
      The Integrated Stress Response (ISR) refers to signaling pathways initiated by stress-activated eIF2‹ kinases. Distinct eIF2‹ kinases respond to different stress signals, including amino acid deprivation and mitochondrial stress. Such stress-induced eIF2‹ phosphorylation attenuates general mRNA translation and, at the same time, stimulates the preferential translation of specific downstream factors to orchestrate an adaptive gene expression program. In recent years, there have been significant new advances in our understanding of ISR during metabolic stress adaptation. Here, I discuss those advances, reviewing among others the ISR activation mechanisms in response to amino acid deprivation and mitochondrial stress. In addition, I review how ISR regulates the amino acid metabolic pathways and how changes in the ISR impact the physiology and pathology of various disease models.
    Keywords:  ATF4; GCN1; GCN2; HRI; Integrated Stress Response; amino acid deprivation; cysteine; eIF2‹; glutathione; mitochondrial stress; serine biosynthesis
    DOI:  https://doi.org/10.1016/j.jbc.2024.107151
  12. Nat Commun. 2024 Mar 11. 15(1): 2207
    Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.
    Jordan Currie, Vyshnavi Manda, Sean K Robinson, Celine Lai, Vertica Agnihotri, Veronica Hidalgo, R W Ludwig, Kai Zhang, Jay Pavelka, Zhao V Wang, June-Wha Rhee, Maggie P Y Lam, Edward Lau.
      The spatial and temporal distributions of proteins are critical to protein function, but cannot be directly assessed by measuring protein bundance. Here we describe a mass spectrometry-based proteomics strategy, Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently protein turnover rates and subcellular localization in the same experiment. Applying the method, we find that unfolded protein response (UPR) has different effects on protein turnover dependent on their subcellular location in human AC16 cells, with proteome-wide slowdown but acceleration among stress response proteins in the ER and Golgi. In parallel, UPR triggers broad differential localization of proteins including RNA-binding proteins and amino acid transporters. Moreover, we observe newly synthesized proteins including EGFR that show a differential localization under stress than the existing protein pools, reminiscent of protein trafficking disruptions. We next applied SPLAT to an induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) model of cancer drug cardiotoxicity upon treatment with the proteasome inhibitor carfilzomib. Paradoxically, carfilzomib has little effect on global average protein half-life, but may instead selectively disrupt sarcomere protein homeostasis. This study provides a view into the interactions of protein spatial and temporal dynamics and demonstrates a method to examine protein homeostasis regulations in stress and drug response.
    DOI:  https://doi.org/10.1038/s41467-024-46600-5
  13. Cancer Res. 2024 Mar 15.
    NCI Cancer Research Data Commons: Cloud-based Analytical Resources.
    David Pot, Zelia Worman, Alexander Baumann, Shirish Pathak, Rowan Beck, Katherine Thayer, Tanja M Davidsen, Erika Kim, Brandi Davis-Dusenbery, John Otridge, Todd Pihl, The Crdc Program, Jill S Barnholtz-Sloan, Anthony R Kerlavage.
      The NCI's Cloud Resources (CRs) are the analytical components of the Cancer Research Data Commons (CRDC) ecosystem. This review describes how the three CRs (Broad Institute FireCloud, Institute for Systems Biology Cancer Gateway in the Cloud, and Seven Bridges Cancer Genomics Cloud) provide access and availability to large, cloud-hosted, multi-modal cancer datasets, as well as offer tools and workspaces for performing data analysis where the data resides, without download or storage. In addition, users can upload their own data and tools into their workspaces, allowing researchers to create custom analysis workflows and integrate CRDC-hosted data with their own.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-23-2657
  14. Cancer Res. 2024 Mar 15.
    NCI Cancer Research Data Commons: Resources to Share Key Cancer Data.
    Zhining Wang, Tanja M Davidsen, Gina R Kuffel, KanakaDurga Addepalli, Amanda Bell, Esmeralda Casas-Silva, Hayley Dingerdissen, Keyvan Farahani, Andrey Fedorov, Sharon Gaheen, Robert L Grossman, Ron Kikinis, Erika Kim, John Otridge, Todd Pihl, Melissa Porter, Henry Rodriguez, Louis M Staudt, Ratna R Thangudu, Sudha Venkatachari, Jean Claude Zenklusen, Xu Zhang, Jill S Barnholtz-Sloan, The Crdc Program, Anthony R Kerlavage.
      Since 2014, the National Cancer Institute (NCI) has launched a series of data commons as part of the Cancer Research Data Commons (CRDC) ecosystem housing genomic, proteomic, imaging, and clinical data to support cancer research and promote data sharing of NCI-funded studies. This review describes each data commons (Genomic Data Commons, Proteomic Data Commons, Integrated Canine Data Commons, Cancer Data Service, Imaging Data Commons, and Clinical and Translational Data Commons), including their unique and shared features, accomplishments, and challenges. Also discussed is how the CRDC data commons implement Findable, Accessible, Interoperable, Reusable (FAIR) principles and promote data sharing in support of the new NIH Data Management and Sharing Policy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-23-2468
  15. Cell Genom. 2024 Mar 13. pii: S2666-979X(24)00060-0. [Epub ahead of print]4(3): 100519
    Modular vector assembly enables rapid assessment of emerging CRISPR technologies.
    Abby V McGee, Yanjing V Liu, Audrey L Griffith, Zsofia M Szegletes, Bronte Wen, Carolyn Kraus, Nathan W Miller, Ryan J Steger, Berta Escude Velasco, Justin A Bosch, Jonathan D Zirin, Raghuvir Viswanatha, Erik J Sontheimer, Amy Goodale, Matthew A Greene, Thomas M Green, John G Doench.
      The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
    Keywords:  AAV; CRISPR; CRISPRi; Cas12a; Cas9; Golden Gate; functional genomics; lentivirus
    DOI:  https://doi.org/10.1016/j.xgen.2024.100519
  16. Commun Biol. 2024 Mar 14. 7(1): 323
    A neural network-based model framework for cell-fate decisions and development.
    Mátyás Paczkó, Dániel Vörös, Péter Szabó, Gáspár Jékely, Eörs Szathmáry, András Szilágyi.
      Gene regulatory networks (GRNs) fulfill the essential function of maintaining the stability of cellular differentiation states by sustaining lineage-specific gene expression, while driving the progression of development. However, accounting for the relative stability of intermediate differentiation stages and their divergent trajectories remains a major challenge for models of developmental biology. Here, we develop an empirical data-based associative GRN model (AGRN) in which regulatory networks store multilineage stage-specific gene expression profiles as associative memory patterns. These networks are capable of responding to multiple instructive signals and, depending on signal timing and identity, can dynamically drive the differentiation of multipotent cells toward different cell state attractors. The AGRN dynamics can thus generate diverse lineage-committed cell populations in a robust yet flexible manner, providing an attractor-based explanation for signal-driven cell fate decisions during differentiation and offering a readily generalizable modelling tool that can be applied to a wide variety of cell specification systems.
    DOI:  https://doi.org/10.1038/s42003-024-05985-1
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