bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024‒01‒28
twenty-one papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. The mTORC2 signaling network: targets and cross-talks.
  2. Integrative modeling uncovers p21-driven drug resistance and prioritizes therapies for PIK3CA-mutant breast cancer.
  3. p300 nucleocytoplasmic shuttling underlies mTORC1 hyperactivation in Hutchinson-Gilford progeria syndrome.
  4. Functional selectivity of Receptor Tyrosine Kinases regulates distinct cellular outputs.
  5. SHMT2 promotes papillary thyroid cancer metastasis through epigenetic activation of AKT signaling.
  6. AKT1 phosphorylation of cytoplasmic ME2 induces a metabolic switch to glycolysis for tumorigenesis.
  7. scPerturb: harmonized single-cell perturbation data.
  8. Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling.
  9. Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.
  10. Mass spectrometry-based proteomics data from thousands of HeLa control samples.
  11. A genetically-encoded fluorescent biosensor for visualization of acetyl-CoA in live cells.
  12. Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution.
  13. CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.
  14. Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.
  15. BRAF - a tumour-agnostic drug target with lineage-specific dependencies.
  16. Anti-correlated feature selection prevents false discovery of subpopulations in scRNAseq.
  17. RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells.
  18. Phosphorylation Codes in IRS-1 and IRS-2 Are Associated with the Activation/Inhibition of Insulin Canonical Signaling Pathways.
  19. Monitoring phosphorylation and acetylation of CRISPR-mediated HiBiT-tagged endogenous proteins.
  20. Deciphering cell states and genealogies of human hematopoiesis.
  21. Time-dependent effects of BRAF-V600E on cell cycling, metabolism, and function in engineered myocardium.
  1. Biochem J. 2024 Jan 25. 481(2): 45-91
    The mTORC2 signaling network: targets and cross-talks.
    Aparna Ragupathi, Christian Kim, Estela Jacinto.
      The mechanistic target of rapamycin, mTOR, controls cell metabolism in response to growth signals and stress stimuli. The cellular functions of mTOR are mediated by two distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. Rapamycin and its analogs are currently used in the clinic to treat a variety of diseases and have been instrumental in delineating the functions of its direct target, mTORC1. Despite the lack of a specific mTORC2 inhibitor, genetic studies that disrupt mTORC2 expression unravel the functions of this more elusive mTOR complex. Like mTORC1 which responds to growth signals, mTORC2 is also activated by anabolic signals but is additionally triggered by stress. mTORC2 mediates signals from growth factor receptors and G-protein coupled receptors. How stress conditions such as nutrient limitation modulate mTORC2 activation to allow metabolic reprogramming and ensure cell survival remains poorly understood. A variety of downstream effectors of mTORC2 have been identified but the most well-characterized mTORC2 substrates include Akt, PKC, and SGK, which are members of the AGC protein kinase family. Here, we review how mTORC2 is regulated by cellular stimuli including how compartmentalization and modulation of complex components affect mTORC2 signaling. We elaborate on how phosphorylation of its substrates, particularly the AGC kinases, mediates its diverse functions in growth, proliferation, survival, and differentiation. We discuss other signaling and metabolic components that cross-talk with mTORC2 and the cellular output of these signals. Lastly, we consider how to more effectively target the mTORC2 pathway to treat diseases that have deregulated mTOR signaling.
    Keywords:  Akt; SIN1; cell growth; mTOR; mTORC2; metabolism; rictor
    DOI:  https://doi.org/10.1042/BCJ20220325
  2. NPJ Precis Oncol. 2024 Jan 26. 8(1): 20
    Integrative modeling uncovers p21-driven drug resistance and prioritizes therapies for PIK3CA-mutant breast cancer.
    Hon Yan Kelvin Yip, Sung-Young Shin, Annabel Chee, Ching-Seng Ang, Fernando J Rossello, Lee Hwa Wong, Lan K Nguyen, Antonella Papa.
      Utility of PI3Kα inhibitors like BYL719 is limited by the acquisition of genetic and non-genetic mechanisms of resistance which cause disease recurrence. Several combination therapies based on PI3K inhibition have been proposed, but a way to systematically prioritize them for breast cancer treatment is still missing. By integrating published and in-house studies, we have developed in silico models that quantitatively capture dynamics of PI3K signaling at the network-level under a BYL719-sensitive versus BYL719 resistant-cell state. Computational predictions show that signal rewiring to alternative components of the PI3K pathway promote resistance to BYL719 and identify PDK1 as the most effective co-target with PI3Kα rescuing sensitivity of resistant cells to BYL719. To explore whether PI3K pathway-independent mechanisms further contribute to BYL719 resistance, we performed phosphoproteomics and found that selection of high levels of the cell cycle regulator p21 unexpectedly promoted drug resistance in T47D cells. Functionally, high p21 levels favored repair of BYL719-induced DNA damage and bypass of the associated cellular senescence. Importantly, targeted inhibition of the check-point inhibitor CHK1 with MK-8776 effectively caused death of p21-high T47D cells, thus establishing a new vulnerability of BYL719-resistant breast cancer cells. Together, our integrated studies uncover hidden molecular mediators causing resistance to PI3Kα inhibition and provide a framework to prioritize combination therapies for PI3K-mutant breast cancer.
    DOI:  https://doi.org/10.1038/s41698-024-00496-y
  3. Nat Cell Biol. 2024 Jan 24.
    p300 nucleocytoplasmic shuttling underlies mTORC1 hyperactivation in Hutchinson-Gilford progeria syndrome.
    Sung Min Son, So Jung Park, Sophia Y Breusegem, Delphine Larrieu, David C Rubinsztein.
      The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.
    DOI:  https://doi.org/10.1038/s41556-023-01338-y
  4. Front Cell Dev Biol. 2023 ;11 1348056
    Functional selectivity of Receptor Tyrosine Kinases regulates distinct cellular outputs.
    Sakim S Samad, Jean-Marc Schwartz, Chiara Francavilla.
      Functional selectivity refers to the activation of differential signalling and cellular outputs downstream of the same membrane-bound receptor when activated by two or more different ligands. Functional selectivity has been described and extensively studied for G-protein Coupled Receptors (GPCRs), leading to specific therapeutic options for dysregulated GPCRs functions. However, studies regarding the functional selectivity of Receptor Tyrosine Kinases (RTKs) remain sparse. Here, we will summarize recent data about RTK functional selectivity focusing on how the nature and the amount of RTK ligands and the crosstalk of RTKs with other membrane proteins regulate the specificity of RTK signalling. In addition, we will discuss how structural changes in RTKs upon ligand binding affects selective signalling pathways. Much remains to be known about the integration of different signals affecting RTK signalling specificity to orchestrate long-term cellular outcomes. Recent advancements in omics, specifically quantitative phosphoproteomics, and in systems biology methods to study, model and integrate different types of large-scale omics data have increased our ability to compare several signals affecting RTK functional selectivity in a global, system-wide fashion. We will discuss how such methods facilitate the exploration of important signalling hubs and enable data-driven predictions aiming at improving the efficacy of therapeutics for diseases like cancer, where redundant RTK signalling pathways often compromise treatment efficacy.
    Keywords:  EGFR; FGFR; cell signalling; functional selectivity; ligand bias; ligand concentration; modelling; receptor tyrosine kinase (RTK)
    DOI:  https://doi.org/10.3389/fcell.2023.1348056
  5. Cell Death Dis. 2024 Jan 25. 15(1): 87
    SHMT2 promotes papillary thyroid cancer metastasis through epigenetic activation of AKT signaling.
    Min Sun, Mingjian Zhao, Ruowen Li, Yankun Zhang, Xiaojia Shi, Changyuan Ding, Chunhong Ma, Jinghui Lu, Xuetian Yue.
      Cancer cells alter their metabolism and epigenetics to support cancer progression. However, very few modulators connecting metabolism and epigenetics have been uncovered. Here, we reveal that serine hydroxymethyltransferase-2 (SHMT2) generates S-adenosylmethionine (SAM) to epigenetically repress phosphatase and tensin homolog (PTEN), leading to papillary thyroid cancer (PTC) metastasis depending on activation of AKT signaling. SHMT2 is elevated in PTC, and is associated with poor prognosis. Overexpressed SHMT2 promotes PTC metastasis both in vitro and in vivo. Proteomic enrichment analysis shows that AKT signaling is activated, and is positively associated with SHMT2 in PTC specimens. Blocking AKT activation eliminates the effects of SHMT2 on promoting PTC metastasis. Furthermore, SHMT2 expression is negatively associated with PTEN, a negative AKT regulator, in PTC specimens. Mechanistically, SHMT2 catalyzes serine metabolism and produces activated one-carbon units that can generate SAM for the methylation of CpG islands in PTEN promoter for PTEN suppression and following AKT activation. Importantly, interference with PTEN expression affects SHMT2 function by promoting AKT signaling activation and PTC metastasis. Collectively, our research demonstrates that SHMT2 connects metabolic reprogramming and epigenetics, contributing to the poor progression of PTC.
    DOI:  https://doi.org/10.1038/s41419-024-06476-1
  6. Nat Commun. 2024 Jan 23. 15(1): 686
    AKT1 phosphorylation of cytoplasmic ME2 induces a metabolic switch to glycolysis for tumorigenesis.
    Taiqi Chen, Siyi Xie, Jie Cheng, Qiao Zhao, Hong Wu, Peng Jiang, Wenjing Du.
      Many types of tumors feature aerobic glycolysis for meeting their increased energetic and biosynthetic demands. However, it remains still unclear how this glycolytic phenomenon is achieved and coordinated with other metabolic pathways in tumor cells in response to growth stimuli. Here we report that activation of AKT1 induces a metabolic switch to glycolysis from the mitochondrial metabolism via phosphorylation of cytoplasmic malic enzyme 2 (ME2), named ME2fl (fl means full length), favoring an enhanced glycolytic phenotype. Mechanistically, in the cytoplasm, AKT1 phosphorylates ME2fl at serine 9 in the mitochondrial localization signal peptide at the N-terminus, preventing its mitochondrial translocation. Unlike mitochondrial ME2, which accounts for adjusting the tricarboxylic acid (TCA) cycle, ME2fl functions as a scaffold that brings together the key glycolytic enzymes phosphofructokinase (PFKL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as Lactate dehydrogenase A (LDHA), to promote glycolysis in the cytosol. Thus, through phosphorylation of ME2fl, AKT1 enhances the glycolytic capacity of tumor cells in vitro and in vivo, revealing an unexpected role for subcellular translocation switching of ME2 mediated by AKT1 in the metabolic adaptation of tumor cells to growth stimuli.
    DOI:  https://doi.org/10.1038/s41467-024-44772-8
  7. Nat Methods. 2024 Jan 26.
    scPerturb: harmonized single-cell perturbation data.
    Stefan Peidli, Tessa D Green, Ciyue Shen, Torsten Gross, Joseph Min, Samuele Garda, Bo Yuan, Linus J Schumacher, Jake P Taylor-King, Debora S Marks, Augustin Luna, Nils Blüthgen, Chris Sander.
      Analysis across a growing number of single-cell perturbation datasets is hampered by poor data interoperability. To facilitate development and benchmarking of computational methods, we collect a set of 44 publicly available single-cell perturbation-response datasets with molecular readouts, including transcriptomics, proteomics and epigenomics. We apply uniform quality control pipelines and harmonize feature annotations. The resulting information resource, scPerturb, enables development and testing of computational methods, and facilitates comparison and integration across datasets. We describe energy statistics (E-statistics) for quantification of perturbation effects and significance testing, and demonstrate E-distance as a general distance measure between sets of single-cell expression profiles. We illustrate the application of E-statistics for quantifying similarity and efficacy of perturbations. The perturbation-response datasets and E-statistics computation software are publicly available at scperturb.org. This work provides an information resource for researchers working with single-cell perturbation data and recommendations for experimental design, including optimal cell counts and read depth.
    DOI:  https://doi.org/10.1038/s41592-023-02144-y
  8. J Biol Chem. 2024 Jan 23. pii: S0021-9258(24)00055-3. [Epub ahead of print] 105679
    Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling.
    Atsushi Morimoto, Nobumasa Takasugi, Yuexuan Pan, Sho Kubota, Naoshi Dohmae, Yumi Abiko, Koji Uchida, Yoshito Kumagai, Takashi Uehara.
      Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as cysteine (Cys) in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed phosphatidylinositol-3 kinase (PI3K)-Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor (EGF) receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K-Akt signaling by MVK reversed EGF-induced negative regulation of autophagy, and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K-Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K-Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.
    Keywords:  autophagy; cell signaling; chemical modification; glucose uptake; phosphatidylinositol 3‐kinase (PI 3‐kinase)
    DOI:  https://doi.org/10.1016/j.jbc.2024.105679
  9. bioRxiv. 2024 Jan 04. pii: 2024.01.03.574067. [Epub ahead of print]
    Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.
    Adam X Miranda, Justin Kemp, Brad Davidson, Sara Erika Bellomo, Verda Agan, Alexandra Manoni, Caterina Marchiò, Sarah Croessmann, Ben H Park, Emily Hodges.
      Background: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype.Results: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases.
    Conclusions: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.
    DOI:  https://doi.org/10.1101/2024.01.03.574067
  10. Sci Data. 2024 Jan 23. 11(1): 112
    Mass spectrometry-based proteomics data from thousands of HeLa control samples.
    Henry Webel, Yasset Perez-Riverol, Annelaura Bach Nielsen, Simon Rasmussen.
      Here we provide a curated, large scale, label free mass spectrometry-based proteomics data set derived from HeLa cell lines for general purpose machine learning and analysis. Data access and filtering is a tedious task, which takes up considerable amounts of time for researchers. Therefore we provide machine based metadata for easy selection and overview along the 7,444 raw files and MaxQuant search output. For convenience, we provide three filtered and aggregated development datasets on the protein groups, peptides and precursors level. Next to providing easy to access training data, we provide a SDRF file annotating each raw file with instrument settings allowing automated reprocessing. We encourage others to enlarge this data set by instrument runs of further HeLa samples from different machine types by providing our workflows and analysis scripts.
    DOI:  https://doi.org/10.1038/s41597-024-02922-z
  11. bioRxiv. 2024 Jan 01. pii: 2023.12.31.573774. [Epub ahead of print]
    A genetically-encoded fluorescent biosensor for visualization of acetyl-CoA in live cells.
    Joseph J Smith, Taylor R Valentino, Austin H Ablicki, Riddhidev Banerjee, Adam R Colligan, Debra M Eckert, Gabrielle A Desjardins, Katharine L Diehl.
      Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA production and consumption are highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. In this work, we engineer an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). We biochemically characterize the sensor and demonstrate its selectivity for acetyl-CoA over other CoA species. We then deploy the biosensor in E. coli and HeLa cells to demonstrate its utility in living cells. In E. coli , we show that the biosensor enables detection of rapid changes in acetyl-CoA levels. In human cells, we show that the biosensor enables subcellular detection and reveals the compartmentalization of acetyl-CoA metabolism.
    DOI:  https://doi.org/10.1101/2023.12.31.573774
  12. Nat Biotechnol. 2024 Jan 25.
    Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution.
    Jason Z Zhang, William H Nguyen, Nathan Greenwood, John C Rose, Shao-En Ong, Dustin J Maly, David Baker.
      The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies.
    DOI:  https://doi.org/10.1038/s41587-023-02107-w
  13. Int J Mol Sci. 2024 Jan 12. pii: 985. [Epub ahead of print]25(2):
    CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.
    Ruba Hammad, Jamal Alzubi, Manuel Rhiel, Kay O Chmielewski, Laura Mosti, Julia Rositzka, Marcel Heugel, Jan Lawrenz, Valentina Pennucci, Birgitta Gläser, Judith Fischer, Axel Schambach, Thomas Moritz, Nico Lachmann, Tatjana I Cornu, Claudio Mussolino, Richard Schäfer, Toni Cathomen.
      The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.
    Keywords:  AAV; CRISPR; Cas12a; Cpf1; Cpf1 Ultra; HSC; HSPC; T cell; genome editing; iPSC
    DOI:  https://doi.org/10.3390/ijms25020985
  14. bioRxiv. 2024 Jan 05. pii: 2024.01.04.574239. [Epub ahead of print]
    Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.
    Ziqing Liu, Natalie T Tanke, Alexandra Neal, Tianji Yu, Tershona Branch, Jean G Cook, Victoria L Bautch.
      Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of proliferative tip cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.
    Keywords:  Cell cycle; G2 arrest; HUVEC; blood vessel; endothelial cells
    DOI:  https://doi.org/10.1101/2024.01.04.574239
  15. Nat Rev Clin Oncol. 2024 Jan 26.
    BRAF - a tumour-agnostic drug target with lineage-specific dependencies.
    Aphrothiti J Hanrahan, Ziyu Chen, Neal Rosen, David B Solit.
      In June 2022, the FDA granted Accelerated Approval to the BRAF inhibitor dabrafenib in combination with the MEK inhibitor trametinib for the treatment of adult and paediatric patients (≥6 years of age) with unresectable or metastatic BRAFV600E-mutant solid tumours, except for BRAFV600E-mutant colorectal cancers. The histology-agnostic approval of dabrafenib plus trametinib marks the culmination of two decades of research into the landscape of BRAF mutations in human cancers, the biochemical mechanisms underlying BRAF-mediated tumorigenesis, and the clinical development of selective RAF and MEK inhibitors. Although the majority of patients with BRAFV600E-mutant tumours derive clinical benefit from BRAF inhibitor-based combinations, resistance to treatment develops in most. In this Review, we describe the biochemical basis for oncogenic BRAF-induced activation of MAPK signalling and pan-cancer and lineage-specific mechanisms of intrinsic, adaptive and acquired resistance to BRAF inhibitors. We also discuss novel RAF inhibitors and drug combinations designed to delay the emergence of treatment resistance and/or expand the population of patients with BRAF-mutant cancers who benefit from molecularly targeted therapies.
    DOI:  https://doi.org/10.1038/s41571-023-00852-0
  16. Nat Commun. 2024 Jan 24. 15(1): 699
    Anti-correlated feature selection prevents false discovery of subpopulations in scRNAseq.
    Scott R Tyler, Daniel Lozano-Ojalvo, Ernesto Guccione, Eric E Schadt.
      While sub-clustering cell-populations has become popular in single cell-omics, negative controls for this process are lacking. Popular feature-selection/clustering algorithms fail the null-dataset problem, allowing erroneous subdivisions of homogenous clusters until nearly each cell is called its own cluster. Using real and synthetic datasets, we find that anti-correlated gene selection reduces or eliminates erroneous subdivisions, increases marker-gene selection efficacy, and efficiently scales to millions of cells.
    DOI:  https://doi.org/10.1038/s41467-023-43406-9
  17. bioRxiv. 2024 Jan 11. pii: 2024.01.09.574940. [Epub ahead of print]
    RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells.
    Vasanth Siruvallur Murali, Divya Rajendran, Tadamoto Isogai, Ralph J DeBerardinis, Gaudenz Danuser.
      Cutaneous melanomas harboring a B-Raf V600E mutation are treated with immune check point inhibitors or kinase inhibitor combination therapies relying on MAPK inhibitors (MAPKi) Dabrafenib and Trametinib (Curti and Faries, 2021). However, cells become resistant to treatments over the timespan of a few months. Resistance to MAPKi has been associated with adoption of an aggressive amoeboid phenotype characterized by elevated RhoA signaling, enhanced contractility and thick cortical filamentous actin (F-actin) structures (Kim et al., 2016; Misek et al., 2020). Targeting active RhoA through Rho-kinase (ROCK) inhibitors, either alone or in combination with immunotherapies, reverts MAPKi-resistance (Misek et al., 2020; Orgaz et al., 2020). Yet, the mechanisms for this behavior remain largely unknown. Given our recent findings of cytoskeleton's role in cancer cell proliferation (Mohan et al., 2019), survival (Weems et al., 2023), and metabolism (Park et al., 2020), we explored possibilities by which RhoA-driven changes in cytoskeleton structure may confer resistance. We confirmed elevated activation of RhoA in a panel of MAPKi-resistant melanoma cell lines, leading to a marked increase in the presence of contractile F-actin bundles. Moreover, these cells had increased glucose uptake and glycolysis, a phenotype disrupted by pharmacological perturbation of ROCK. However, glycolysis was unaffected by disruption of F-actin bundles, indicating that glycolytic stimulation in MAPKi-resistant melanoma is independent of F-actin organization. Instead, our findings highlight a mechanism in which elevated RhoA signaling activates ROCK, leading to the activation of insulin receptor substrate 1 (IRS1) and P85 of the PI3K pathway, which promotes cell surface expression of GLUT1 and elevated glucose uptake. Application of ROCK inhibitor GSK269962A results in reduced glucose uptake and glycolysis, thus impeding cell proliferation. Our study adds a mechanism to the proposed use of ROCK inhibitors for long-term treatments on MAPKi-resistant melanomas.
    DOI:  https://doi.org/10.1101/2024.01.09.574940
  18. Curr Issues Mol Biol. 2024 Jan 09. 46(1): 634-649
    Phosphorylation Codes in IRS-1 and IRS-2 Are Associated with the Activation/Inhibition of Insulin Canonical Signaling Pathways.
    Anabel Martínez Báez, Guadalupe Ayala, Adolfo Pedroza-Saavedra, Hilda M González-Sánchez, Lilia Chihu Amparan.
      Insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) are signaling adaptor proteins that participate in canonical pathways, where insulin cascade activation occurs, as well as in non-canonical pathways, in which phosphorylation of substrates is carried out by a diverse array of receptors including integrins, cytokines, steroid hormones, and others. IRS proteins are subject to a spectrum of post-translational modifications essential for their activation, encompassing phosphorylation events in distinct tyrosine, serine, and threonine residues. Tyrosine residue phosphorylation is intricately linked to the activation of the insulin receptor cascade and its interaction with SH2 domains within a spectrum of proteins, including PI3K. Conversely, serine residue phosphorylation assumes a different function, serving to attenuate the effects of insulin. In this review, we have identified over 50 serine residues within IRS-1 that have been reported to undergo phosphorylation orchestrated by a spectrum of kinases, thereby engendering the activation or inhibition of different signaling pathways. Furthermore, we delineate the phosphorylation of over 10 distinct tyrosine residues at IRS-1 or IRS-2 in response to insulin, a process essential for signal transduction and the subsequent activation of PI3K.
    Keywords:  IRS-1; IRS-2; insulin; phosphorylation
    DOI:  https://doi.org/10.3390/cimb46010041
  19. Sci Rep. 2024 Jan 25. 14(1): 2138
    Monitoring phosphorylation and acetylation of CRISPR-mediated HiBiT-tagged endogenous proteins.
    Juliano Alves, Marie Schwinn, Thomas Machleidt, Said A Goueli, James J Cali, Hicham Zegzouti.
      Intracellular pathways transduce signals through changes in post-translational modifications (PTMs) of effector proteins. Among the approaches used to monitor PTM changes are immunoassays and overexpression of recombinant reporter genes. Genome editing by CRISPR/Cas9 provides a new means to monitor PTM changes by inserting reporters onto target endogenous genes while preserving native biology. Ideally, the reporter should be small in order not to interfere with the processes mediated by the target while sensitive enough to detect tightly expressed proteins. HiBiT is a 1.3 kDa reporter peptide capable of generating bioluminescence through complementation with LgBiT, an 18 kDa subunit derived from NanoLuc. Using HiBiT CRISPR/Cas9-modified cell lines in combination with fluorescent antibodies, we developed a HiBiT-BRET immunoassay (a.k.a. Immuno-BRET). This is a homogeneous immunoassay capable of monitoring post-translational modifications on diverse protein targets. Its usefulness was demonstrated for the detection of phosphorylation of multiple signaling pathway targets (EGFR, STAT3, MAPK8 and c-MET), as well as chromatin containing histone H3 acetylation on lysine 9 and 27. These results demonstrate the ability to efficiently monitor endogenous biological processes modulated by post-translational modifications using a small bioluminescent peptide tag and fluorescent antibodies, providing sensitive quantitation of the response dynamics to multiple stimuli.
    DOI:  https://doi.org/10.1038/s41598-024-51887-x
  20. Nature. 2024 Jan 22.
    Deciphering cell states and genealogies of human hematopoiesis.
    Chen Weng, Fulong Yu, Dian Yang, Michael Poeschla, L Alexander Liggett, Matthew G Jones, Xiaojie Qiu, Lara Wahlster, Alexis Caulier, Jeffrey A Hussmann, Alexandra Schnell, Kathryn E Yost, Luke Koblan, Jorge D Martin-Rufino, Joseph Min, Alessandro Hammond, Daniel Ssozi, Raphael Bueno, Hari Mallidi, Antonia Kreso, Javier Escabi, William M Rideout, Tyler Jacks, Sahand Hormoz, Peter van Galen, Jonathan S Weissman, Vijay G Sankaran.
      The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived hematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human hematopoiesis and how this changes with age remain incompletely understood. While recent insights have emerged from barcoding studies in model systems4,5,16,17, simultaneous detection of cell states and phylogenies from natural barcodes in humans has been challenging. Here, we introduce an improved single-cell lineage tracing system based on deep detection of naturally-occurring mitochondrial DNA (mtDNA) mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as differences in total HSC output as well as biases toward the production of different mature cell types. We also find that the diversity of HSC clones decreases dramatically with age leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides the first clonally-resolved and cell-state aware atlas of human hematopoiesis at single-cell resolution revealing an unappreciated functional diversity of human HSC clones and more broadly paves the way for refined studies of clonal dynamics across a range of tissues in human health and disease.
    DOI:  https://doi.org/10.1038/s41586-024-07066-z
  21. Sci Adv. 2024 Jan 26. 10(4): eadh2598
    Time-dependent effects of BRAF-V600E on cell cycling, metabolism, and function in engineered myocardium.
    Nicholas Strash, Sophia DeLuca, Geovanni L Janer Carattini, Yifan Chen, Tianyu Wu, Abbigail Helfer, Jacob Scherba, Isabella Wang, Mehul Jain, Ramona Naseri, Nenad Bursac.
      Candidate cardiomyocyte (CM) mitogens such as those affecting the extracellular signal-regulated kinase (ERK) signaling pathway represent potential targets for functional heart regeneration. We explored whether activating ERK via a constitutively active mutant of B-raf proto-oncogene (BRAF), BRAF-V600E (caBRAF), can induce proproliferative effects in neonatal rat engineered cardiac tissues (ECTs). Sustained CM-specific caBRAF expression induced chronic ERK activation, substantial tissue growth, deficit in sarcomeres and contractile function, and tissue stiffening, all of which persisted for at least 4 weeks of culture. caBRAF-expressing CMs in ECTs exhibited broad transcriptomic changes, shift to glycolytic metabolism, loss of connexin-43, and a promigratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid and precedes functional decline, and the effects are reversible only with short-lived ERK activation. Together, direct activation of the BRAF kinase is sufficient to modulate CM cycling and functional phenotype, offering mechanistic insights into roles of ERK signaling in the context of cardiac development and regeneration.
    DOI:  https://doi.org/10.1126/sciadv.adh2598
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